CN101415819A - Anti-IL-6 antibodies, compositions, methods and uses - Google Patents

Anti-IL-6 antibodies, compositions, methods and uses Download PDF

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CN101415819A
CN101415819A CNA2006800240371A CN200680024037A CN101415819A CN 101415819 A CN101415819 A CN 101415819A CN A2006800240371 A CNA2006800240371 A CN A2006800240371A CN 200680024037 A CN200680024037 A CN 200680024037A CN 101415819 A CN101415819 A CN 101415819A
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seq
antibody
nos
nucleotide sequence
aminoacid sequence
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CN101415819B (en
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Y·陈
D·加德纳
D·M·克赖特
M·W·拉克
B·梁
D·J·希利
X·-Y·R·宋
V·斯托贾诺韦-苏苏里
R·W·斯威特
S·H·谭
S·-J·吴
J·杨
D·M·马奎斯
E·M·史密斯
A·P·瓦塞罗特
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Janssen Biotech Inc
Applied Molecular Evolution Inc
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Centocor Inc
Applied Molecular Evolution Inc
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Abstract

An anti-IL-6 antibody, includes isolated nucleic acids that encode at least one anti-IL-6 antibody, vectors, host cells, transgenic animals or plants, and methods of making and using thereof have applications in diagnostic and/or therapeutic compositions, methods and devices.

Description

Anti-IL-6 antibodies, composition, method and application
Invention field
The present invention relates to be specific at least a IL-6 albumen or its segmental antibody that comprises specific part or variant, and the nucleic acid of the antiidiotypic antibody and the above-mentioned anti-IL-6 antibodies of encoding, complementary nucleic acid, carrier, host cell, and preparation and using method, comprise treatment prescription, administration and device.
Background of invention
IL-6 is a kind of by generation of various kinds of cell type and excretory pleiotropy pro-inflammatory cytokine, it should be noted that antigen presenting cell, T and B cell most.IL-6 relates to different activities, for example growth of B cell and differentiation, T cell activation, hemopoiesis, osteoclast activation, keratinocyte growth, neure growth and liver cell activation.IL-6 is in conjunction with striding film or solubility IL-6R and passing through to be the common gp130 conducted signal of some other cytokines.
As shown in systemic lupus erythematosus, multiple myeloma and lymphoproliferative disorder, IL-6 plays an important role in the B cellular abnormality.Similarly, IL-6 also relates to such as the autoimmunity of rheumatoid arthritis and osteoarthritis and the pathogeny of inflammatory diseases.Recently, circumstantial evidence hint IL-6 is relevant with the insulin resistance in chronic obstructive pulmonary disease and the type ii diabetes.IL-6 has proinflammatory and anti-inflammatory action in immunity system, shows that this cytokine may play a major role in the physiological response of regulating disease.Therefore, can provide the treatment benefit potentially at multiple disease field target IL-6.
In multiple disease, observed the increase that IL-6 produces, having comprised: Alzheimer, autoimmune disorder, inflammation, myocardial infarction, osteitis deformans, osteoporosis, solid tumor (renal cell carcinoma), prostate cancer and bladder cancer, nervous system cancer and B-cell malignancies (as CastelemanShi disease, some lymphoma, lymphocytic leukemia and multiple myeloma) such as rheumatoid arthritis.Studies show that IL-6 is relevant with the pathogeny of many these diseases of kind, particularly the pathogeny with cancer is relevant, and therefore blocking IL-6 should be converted into clinical benefit.
Mouse, chimeric and other inhuman anti-IL-6 antibodies have been developed; Yet they may be subject to its effectiveness, validity, often may trigger and unacceptable immunne response (being immunogenicity) and/or need high dosage (referring to Trikha etc., Clin.Can.Res.9,4653-4665, Oct.2003 is incorporated herein by reference at this).For example, the antibody that contains inhuman part usually produces immunne response in the people.Therefore, repetitive administration antibody also is not suitable as treatment, and the removing antibody from circulation of immunocomplex mediation can reduce the effectiveness/validity of antibody.Serum sickness and anaphylaxis are two kinds of caused representative illnesss of antibody that have inhuman part owing to repetitive administration.In this, need have less immunogenicity possibility, promptly the people is more tolerable, and more effective more precisely anti-IL-6 antibodies makes and compare with previous employed anti-IL-6 antibodies that needed dosage is littler.
Summary of the invention
The invention provides isolating people's through engineering approaches anti-IL-6 antibodies (being also referred to as IL-6 antibody), immunoglobulin (Ig), its fragment, cleaved products and other specific parts and variant, and anti-IL-6 antibodies composition, the IL-6 antiidiotypic antibody, their method of coding nucleic acid or complementary nucleic acid, carrier, host cell, composition, prescription, device, transgenic animal, transgenic plant and preparation and use.
On the one hand, the invention provides the isolated nucleic acid molecule that comprises or hybridization complementary with the polynucleotide of specific anti-IL-6 antibodies of coding or antiidiotypic antibody, described antibody comprises at least a its particular sequence, structural domain, part or variant.The present invention further provides the recombinant vectors that comprises described anti-IL-6 antibodies nucleic acid molecule, the host cell that contains above-mentioned nucleic acid and/or recombinant vectors, and the method for preparing and/or utilize above-mentioned antibody, nucleic acid, carrier and/or host cell.
The present invention also provides at least a method that is used for expressing at host cell at least a anti-IL-6 antibodies or IL-6 antiidiotypic antibody, be included in wherein at least a anti-IL-6 antibodies with can detect and/or condition that callable scale reaches under cultivate host cell described herein.
The present invention also provides at least a composition, comprises (a) isolating anti-IL-6 antibodies coding nucleic acid and/or antibody described herein; (b) that be fit to and/or pharmaceutically acceptable carrier or thinner.
The present invention further provides at least a in cell, tissue, organ, animal or patient and/or known in the art and/or described herein related disorders is arranged before, afterwards or during, be used for the administering therapeutic significant quantity to regulate or to treat the anti-IL-6 antibodies method or the composition of at least a illness relevant with IL-6.
The present invention also provides the composition according at least a anti-IL-6 antibodies of the present invention, device and/or the method for at least a delivery treatments or prevention significant quantity.
The present invention further provides at least a in cell, tissue, organ, animal or patient and/or known in the art and/or described herein related disorders is arranged before, afterwards or during, be used to diagnose the anti-IL-6 antibodies method or the composition of at least a illness relevant with IL-6.
The present invention also provides at least a delivering compositions, device and/or the method that is used for according at least a anti-IL-6 antibodies diagnosis of the present invention.
A kind of medical treatment device also is provided, comprise at least a isolating Mammals anti-IL-6 antibodies of the present invention, wherein this device is suitable for contact or uses at least a anti-IL-6 antibodies, IL-6 antiidiotypic antibody, nucleic acid molecule, compound, albumen and/or composition.
Also provide a kind of product that is used for human medicine or diagnostic use, comprise wrapping material and the container that contains at least a isolating anti-IL-6 antibodies of the present invention of solution or lyophilized form.This product can randomly have the container as delivery apparatus or system's integral part.
All inventions described here have been the present invention further provides.
The accompanying drawing summary
Fig. 1 has shown combining of people's engineering and chimeric anti-IL-6 antibodies and IL-6/IL-6R mixture.
Fig. 2 shown people's through engineering approaches anti-IL-6 antibodies of the present invention in conjunction with epi-position.
Fig. 3 has shown that people's through engineering approaches anti-IL-6 antibodies of measuring by ELISA suppresses the MCP-1 secretion of the U937 cell of IL-6 stimulation.
Fig. 4 has shown that people's through engineering approaches anti-IL-6 antibodies of measuring by ELISA suppresses the SAA secretion of the HepG2 cell of IL-6 and IL-1 β stimulation.
Fig. 5 A and 5B have shown the stat3 phosphorylation by people's through engineering approaches anti-IL-6 antibodies blocking-up IL-6 mediation of measuring via the western blot analysis shown in the gel electrophoresis.
Fig. 6 has shown that people's engineering and chimeric anti-IL-6 antibodies suppress people IL-6-inductive SAA generation in the Balb/C mouse.
Fig. 7 has shown that anti-IL-6mAb suppresses the generation of anti-dsDNA autoantibody in NZB/W F1 mouse.
Fig. 8 A has shown and has existed or do not exist under people's through engineering approaches anti-IL-6 antibodies that IL-6 is to the influence of insulin-induced Akt phosphorylation.
Fig. 8 B has shown and has existed or do not exist under people's through engineering approaches anti-IL-6 antibodies that IL-6 is to the western blot analysis of the influence of insulin-induced Akt phosphorylation.
Fig. 9 has shown that the ELISA described in the embodiment 3 is in conjunction with measurement result.
Figure 10 has shown the antiproliferative measurement result of the use IL-6 dependent cells system described in the embodiment 3.
Figure 11 A has shown PI3 kinase activation in the rat hepatocytes of handling with Regular Insulin, IL-6 albumen and anti-IL-6 antibodies.
Figure 11 B has shown the contrast that is used to study the PI3 kinase activation in rat hepatocytes.
Figure 12 A has shown the influence of signal conduction in the IL-6 pair of rat hepatocytes relevant with insulin-induced IR phosphorylation.
Figure 12 B has shown the influence of signal conduction in the IL-6 pair of rat hepatocytes relevant with insulin-induced Akt phosphorylation.
Figure 13 A has shown at the glucose level of handling with anti-IL-6 antibodies in the DIO mouse of back.
Figure 13 B has shown at the insulin level of handling with anti-IL-6 antibodies in the DIO mouse of back.
Figure 13 C has shown at steady-state model evaluation (HOMA) index of handling with anti-IL-6 antibodies in the DIO mouse of back.
Figure 14 A-F has shown the lipid level before and after handling with anti-IL-6 antibodies.
Figure 15 has shown for intraperitoneal glucose tolerance test (ipGTT), handles the timetable of mouse with anti-IL-6mAb.
Detailed Description Of The Invention
The invention provides separation, anti-IL-6 people's engineered antibody restructuring and/or synthetic and for its IL-6 anti-idiotype, and composition and comprise the coding nucleic acid molecule of the polynucleotides of at least a anti-IL-6 antibodies of at least a coding or anti-idiotype. The present invention further includes, but are not limited to prepare and use the method for above-mentioned nucleic acid and antibody and anti-idiotype, comprises diagnosis and therapeutic combination, method and apparatus.
" anti-IL-6 antibodies ", " IL-6 antibody ", " anti-IL-6 antibodies part " or " anti-IL-6 antibodies fragment " and/or " anti-IL-6 antibodies variant " etc. comprise any albumen or peptide that contains the molecule of the immunoglobulin molecules that comprises at least a portion as used herein, such as but not limited at least one complementarity-determining region (CDR) or its ligand binding moiety, heavy chain or variable region of light chain, heavy chain or constant region of light chain, framework region or its any part or IL-6 acceptor or protein-bonded at least a portion of heavy chain or light chain, it can mix in the antibody of the present invention. Such antibody randomly further affects ligands specific, such as but not limited to such antibody in external, original position and/or body, regulate, reduce, increase, antagonism, exciting, alleviate, alleviate, block, suppress, eliminate and/or disturb at least a IL-6 active in conjunction with or the situation of IL-6 receptor active or combination. As nonrestrictive example, the anti-IL-6 antibodies that the present invention is fit to, specific part or variant can be in conjunction with at least a IL-6 molecule or its specific part, variant or domains. The anti-IL-6 antibodies, specific part or the variant that are fit to also optionally affect at least a IL-6 activity or function, and synthetic such as but not limited to RNA, DNA or albumen, IL-6 discharges, the conduction of IL-6 receptor signal, film IL-6 cutting, IL-6 is active, and IL-6 produces and/or is synthetic.
Term " antibody " further is intended to comprise antibody, digestion fragment, specific part and variant thereof, comprises antibody analog or comprises analog antibody or its specific fragment or the structure of part and/or the antibody moiety of function, comprises single-chain antibody and fragment thereof. Functional fragment comprises the Fab in conjunction with mammal IL-6. The Fab that for example, can include but not limited in conjunction with the antibody fragment of IL-6 or its part to contain for the present invention (as by papain digestion), Fab ' (as by pepsin digestion and partial reduction) and F (ab ')2(as by pepsin digestion), facb (as by fibrinolysin digestion), pFc ' (as by pepsin or fibrinolysin digestion), Fd (as by pepsin digestion, partial reduction and reaggregation), Fv or scFv (as producing by Protocols in Molecular Biology) fragment are (referring to such as Colligan, Immunology, the same).
Can utilize enzyme known in the art and/or described herein cutting, synthetic or recombinant technique to produce above-mentioned fragment. Also can utilize the antibody gene that wherein one or more terminator codons is imported natural termination site upstream to produce the antibody of multiple clipped form. For example, can design coding F (ab ')2The combination gene of heavy chain part is to comprise coding CH1The dna sequence dna of district and/or heavy chain hinge area. Can pass through the different antibody moiety of routine techniques chemical crosslinking, maybe can utilize technique for gene engineering different antibody moieties to be prepared as the albumen of adjacency.
Term " people's engineered antibody " is a kind of have complete at least people's framework region and constant region (C as used hereinL、C HThe district is (such as C H1、C H2、C H3) and hinge area) and the antibody that derives from the CDRs of antigen binding antibody. It is the framework region of sequence and the sequence with somatic mutation that complete people's framework region comprises corresponding to ethnic group. CDRs can derive from one or more CDRs in conjunction with IL-6 in any antibody framework region of context. For example, the CDRs of people's engineered antibody of the present invention can derive from context mouse antibodies framework region the CDRs in conjunction with IL-6, then by through engineering approaches with in the complete people's framework region of context in conjunction with IL-6. Usually, people's engineered antibody is essentially no immunogenic in the people.
Similarly, called after primate (monkey, baboon, chimpanzee etc.), rodent (mouse, rat, rabbit, cavy, hamster etc.) and other mammiferous antibody refer to above-mentioned kind, subgenus, genus, subfamily and section's specific antibody. In addition, chimeric antibody can comprise any above-mentioned combination. With respect to the antibody of unmodified, such change or change randomly and preferably keeps or reduces immunogenicity in people or other species. Therefore, people's engineered antibody is different from chimeric or humanized antibody.
It is to be noted can be by the people of energy expressive function rearrangement or non-human animal or protokaryon or the eukaryotic generation people engineered antibody of people's engineering immunoglobulin (Ig) (such as heavy chain and/or light chain) gene. In addition, when people's engineered antibody was single-chain antibody, it can comprise non-existent connection peptide in the natural human antibody. For example, Fv can comprise the connection peptide, as connect variable region of heavy chain and variable region of light chain 2-Yue 8 glycine or other amino acid residues. Such connection peptide is considered to the people source.
Also can use the bispecific, heterospecific, xenogenesis coupling (heteroconjugate) of monoclonal, preferred people, people's engineering or humanized antibody or similar antibody, these antibody have at least two kinds of antibody of the binding specificity of synantigen not. Hereat, a kind of binding specificity is at least a IL-6 albumen, and another kind of binding specificity is then for any other antigen. Method for the preparation of bispecific antibody is known in the art. Traditionally, produce bispecific antibody based on two right coexpression restructuring of heavy chain immunoglobulin-light chain, wherein two heavy chains have not homospecificity (Milstein and Cuello, Nature 305:537 (1983)). Because the random combine of heavy chain immunoglobulin and light chain, these hybridomas (quadroma (quadromas)) produce the mixture of 10 kinds of possible different antibodies molecules, only have a kind of mixture to have correct bispecific structure. Usually carry out the purifying of correct molecule by the affinity chromatography step. Similarly method is disclosed in such as WO 93/08829, the US patent No. 6210668,6193967,6132992,6106833,6060285,6037453,6010902,5989530,5959084,5959083,5932448,5833985,5821333,5807706,5643759,5601819,5582996,5496549,4676980, WO 91/00360, WO 92/00373, EP 03089, Traunecker etc., EMBO is (1991) J.10:3655, Suresh etc., among the Methods in Enzymology 121:210 (1986), each comfortable this is incorporated herein by reference fully.
The anti-IL-6 antibodies that is used for the inventive method and composition optionally have be combined with the IL-6 high-affinity and, optional also preferably have a hypotoxic characteristic. Particularly, for example variable region, constant region and framework region of single component wherein, individually and/or jointly, optional and preferably to have the antibody of the present invention of reduced immunogenicity, specific fragment or variant be useful in the present invention. The antibody that can be used among the present invention randomly has the characteristic that there is the ability of measurable remission and low and/or acceptable toxicity continued treatment patient a period of time. Low or acceptable immunogenicity and/or high-affinity and other characteristics that is fit to can help the treatment results that obtains. Be defined as in the patient with the anti-IL-6 antibodies treatment at this " reduced immunogenicity ", for anti-IL-6 antibodies, the incidence of the antibody of titratable level is present in and is less than 25% use among the patient of the dosage treatment of recommending for the course of disease of recommending during treating, and preferably is present in to be less than among 10% the patient.
The nucleic acid that the present invention separates can be used for producing at least a anti-IL-6 antibodies or its specific variants, described at least a anti-IL-6 antibodies or its specific variants can be used in cell, tissue, organ or animal (comprising mammal and people) vacuum metrics or work with at least a IL-6 illness, be selected from but be not limited at least a immune disorders or disease, cardiovascular disorder or disease, infectiousness, pernicious and/or nervous disorders or disease, or other known or specific IL-6 related disorders play diagnosis, monitoring, adjusting, treatment, alleviation, help and prevent the incidence of disease or mitigation symptoms.
Such method can comprise that the composition that comprises at least a anti-IL-6 antibodies of using effective dose or pharmaceutical composition are in needing such adjusting, treatment, alleviation in symptom, effect or mechanism, prevent or alleviate cell, tissue, organ, animal or patient. Effective dose can comprise about 0.001-500mg/kg/ single (as injecting), repeatedly or the amount of continuous administration, or reach 0.01-5000 μ g/ml serum-concentration/singles, repeatedly or the serum-concentration of continuous administration, maybe when using known method to use and measure, wherein any effective range or value, described method is method described herein or that association area is known.
Antibody of the present invention-generation and formation
Can be randomly clone population by clone well-known in the art, blended clone, immortalized cell or immortalized cell produce at least a anti-IL-6 antibodies of the present invention.Referring to as Ausubel, etc., Current Protocols in Molecular Biology, John Wiley ﹠amp compiled; Sons, Inc., NY, NY (1987-2001); Sambrook, etc., Molecular Cloning:A Laboratory Manual, the 2nd edition, ColdSpring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, etc., Current Protocols inImmunology, John Wiley ﹠amp compiled; Sons, Inc., NY (1994-2001); Colligan etc., CurrentProtocols in Protein Science, John Wiley ﹠amp; Sons, NY, NY, (1997-2001).
Can produce and be specific to people IL-6 albumen or its segmental people's engineered antibody at for example isolating IL-6 albumen of suitable immunogenicity antigen and/or its part (comprising synthetic molecules, for example synthetic peptide).Can produce other special or general Mammals antibody similarly.Can utilize any suitable technology to prepare immunogenicity antigen and generation monoclonal antibody.
In one approach, by the immortalized cell that will be fit to is (as myeloma cell line, such as but not limited to Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, L243, P3X63Ag8.653, Sp2SA3, Sp2MAI, Sp2SS1, Sp2SA5, U937, MLA144, ACTIV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH3T3, HL-60, MLA144, NAMALWA, NEURO 2A etc., or heterozygosis myelomatosis (heteromyelomas), its fusion product, or any cell or fused cell that is derived from wherein, or clone that any other is fit to known in the art) (referring to as www.atcc.org, www.lifetech.com etc.) merge the preparation hybridoma with antibody produced cell, described antibody produced cell is such as but not limited to the spleen isolating or clone, peripheral blood, lymph, tonsilla cell or other immunocytes or contain the cell of B cell, or any other is as cell endogenous or that heterologous nucleic acids is expressed heavy chain or constant region of light chain or variable region or framework region or CDR sequence, described endogenous or heterologous nucleic acids is as reorganization or endogenous virus, bacterium, algae, prokaryotic organism, Amphibians, insect, Reptilia, fish, Mammals, rodent, horse, sheep (ovine), goat, sheep (sheep), primate, eucaryon animal gene group DNA, cDNA, rDNA, Mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, the strand body, duplex or triplex, heterozygote etc. or any their combination.Referring to as Ausubel, the same and Colligan, Immunology, the same, the 2nd chapter is incorporated herein by reference fully at this.
Antibody produced cell also can obtain from already with the peripheral bloods of the people of purpose antigen immune or other suitable animals or preferably obtain from spleen or lymphoglandula.Any other host cell that is fit to also can be used for expressing the allos or the endogenous nucleic acid of coding antibody of the present invention, its specific fragment or variant.Can utilize selectivity culture condition or other currently known methods that is fit to separation and pass through limiting dilution or cell sorting or other currently known methodss clone's fused cell (hybridoma) or reconstitution cell.Can select to produce cell by the assay method (as ELISA) that is fit to the specific antibody of expectation.
Also can use and be used for through engineering approaches or humanization is inhuman or the method for people's antibody and well-known as this area.Humanization or engineered antibody can have one or more inhuman sources amino acid, as but be not limited to mouse, rat, rabbit, non-human primate or other Mammalss.These inhuman amino-acid residues are replaced by the residue that is commonly referred to " input (import) " residue, and it obtains " input " variable region, constant region or other zones from known human sequence usually.
Known people Ig sequence is disclosed in, as www.ncbi.nlm.nih.gov/entrez/query.fcgi; Www.ncbi.nih.gov/igblast; Www.atcc.org/phage/hdb.html; Www.mrc-cpe.cam.ac.uk/ALIGNMENTS.php; Www.kabatdatabase.com/top.html; Ftp.ncbi.nih.gov/repository/kabat; Www.sciquest.com; Www.abcam.com; Www.antibodyresource.com/onlinecomp.html; Www.public.iastate.edu/～pedro/research_tools.html; Www.whfreeman.com/immunology/CH05/kuby05.htm; Www.hhmi.org/grants/lectures/1996/vlab; Www.path.cam.ac.uk/～mrc7/mikeimages.html; Mcb.harvard.edu/BioLinks/Immunology.html; Www.imumunologylink.com; Pathbox.wustl.edu/～hcenter/index.html; Www.appliedbiosystems.com; Www.nal.usda.gov/awic/pubs/antibody; Www.m.ehime-u.ac.jp/～yasuhito/Elisa.html; Www.biodesign.com; Www.cancerresearchuk.org; Www.biotech.ufl.edu; Www.isac-net.org; Baserv.uci.kun.nl/～jraats/links1.html; Www.recab.uni-hd.de/immuno.bme.nwu.edu; Www.mrc-cpe.cam.ac.uk; Www.ibt.unam.mx/vir/V_mice.html; Http:// www.bioinf.org.uk/abs; Antibody.bath.ac.uk; Www.unizh.ch; Www.cryst.bbk.ac.uk/～ubcg07s; Www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.html; Www.path.cam.ac.uk/～mrc7/humanisation/TAHHP.html; Www.ibt.unam.mx/vir/structure/stat_aim.html; Www.biosci.missouri.edu/smithgp/index.html; Www.jerini.de; Kabat et al., SequencesofProteins of Immunological Interest, U.S.Dept.Health (1983), each comfortable this is incorporated herein by reference fully.
Such list entries can be used for reducing immunogenicity or minimizing, increase or changes combination, avidity, association rate (on-rate), dissociation rate (off-rate), avidity, specificity, transformation period or any other suitable characteristic known in the art.Generally speaking, the CDR residue is directly or the most substantially to relate to influencing the antigen combination.Therefore, when the non-human sequence of variable region or constant region can be people or other amino acid and replaces, retaining part or whole inhuman or people CDR sequences.
Antibody also can keep antigenic high-affinity and other favourable biological characteristicses simultaneously randomly by humanization or through engineering approaches or people's antibody engineeringization.For realizing this purpose, can be randomly by utilizing the three-dimensional model of parent, through engineering approaches and humanization sequence, the humanization on analysis parental array and the different concepts and the method for through engineering approaches product prepare humanization (or people) or engineered antibody.Three-dimensional immunoglobulin (Ig) model is usually obtainable and is familiar with by those skilled in the art.Can obtain diagram and show the computer program of the general three-dimensional conformation structure of selected candidate's immunoglobulin sequences.The inspection of these displaying contents is made analysis to residue possible effect in candidate's immunoglobulin sequences function, i.e. the residue analysis that influences candidate's immunoglobulin (Ig) and its antigen bonded ability is carried out.Like this, can select and built up construction district (FR) residue by total and list entries, so that obtain the antibody characteristic of expectation, for example the target antigen avidity of Zeng Jiaing.
In addition, can to comprise ethnic group be the light chain framework region to people's engineering IL-6 antibody of the present invention.In specific embodiment, the light chain kind is that sequence is selected from people VK sequence, includes but not limited to A1, A10, A11, A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, L1, L10, L11, L12, L14, L15, L16, L18, L19, L2, L20, L22, L23, L24, L25, L4/18a, L5, L6, L8, L9, O1, O11, O12, O14, O18, O2, O4, and O8.In some embodiments, the light chain ethnic group is that framework region is selected from V1-11, V1-13, V1-16, V1-17, V1-18, V1-19, V1-2, V1-20, V1-22, V1-3, V1-4, V1-5, V1-7, V1-9, V2-1, V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2-7, V2-8, V3-2, V3-3, V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4, and V5-6.Referring to 2005/005604 couple of PCT WO not of the same race be the description of sequence.
In other embodiment, it is the heavy chain framework region that people's engineering IL-6 antibody of the present invention can comprise ethnic group.In specific embodiment, the heavy chain ethnic group is that framework region is selected from: VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1, and VH7-81.Referring to PCT WO2005/005604 to not of the same race be the description of sequence.
In specific embodiment, variable region of light chain and/or variable region of heavy chain comprise the framework region (as containing 2 or 3 subprovinces, for example FR2 and FR3) of framework region or at least a portion.In some embodiments, FRL1, FRL2, FRL3 or FRL4 are the people fully at least.In other embodiment, FRH1, FRH2, FRH3 or FRH4 are the people fully at least.In certain embodiments, FRL1, FRL2, FRL3 or FRL4 are that kind is sequence (as ethnic group system) or comprises the people's consensus sequence (source that is easy to obtain from above-mentioned known people Ig sequence is located) that is used for specific framework region at least.In other embodiment, FRH1, FRH2, FRH3 or FRH4 are that kind is sequence (as ethnic group system) or comprises the people's consensus sequence that is used for specific framework region at least.In preferred embodiments, framework region is people's framework region (the people's framework regions as shown in following table 13 and 14).In certain embodiments, framework region comprises SEQ ID NOS:105,106,107,108,109,110,111,112 or its combination.
Can use any known method to carry out the humanization or the through engineering approaches of antibody of the present invention, such as but not limited to those methods as described below: Winter (Jones etc., Nature 321:522 (1986); Riechmann etc., Nature 332:323 (1988); Verhoeyen etc., Science 239:1534 (1988)), Sims etc., J.Immunol.151:2296 (1993); Chothia and Lesk, J.Mol.Biol.196:901 (1987), Carter etc., Proc.Natl.Acad.Sci.U.S.A.89:4285 (1992); Presta etc., J.Immunol.151:2623 (1993), the US patent No.: 5723323,5976862,5824514,5817483,5814476,5763192,5723323,5,766886,5714352,6204023,6180370,5693762,5530101,5585089,5225539; 4816567, PCT/:US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP229246, the reference that comprises wherein being quoted, each comfortable this is incorporated herein by reference fully.
In some embodiments, antibody comprises change (as sudden change) Fc district.For example, in certain embodiments, changed the Fc district to reduce or to increase the antibody mediated effect subfunction.In certain embodiments, the Fc district is the isotype that is selected from IgM, IgA, IgG, IgE, or other isotypes.
Select a ground or in addition, can utilize the C1q that changes IL-6 binding molecule Fc district in conjunction with and/or to rely on the combination with one or more additional amino acids modifications of cytotoxicity (CDC) function of complement amino acid modified.Initial specific purposes polypeptide can be in conjunction with C1q and show the Cytotoxic polypeptide that relies on complement.Can modify in conjunction with polypeptide active, that randomly further have a mediation CDC ability having the C1q that is pre-existing in, make that these active one or both are strengthened.Amino acid modified for example being described among the WO0042072 that changes C1q and/or regulate its complement dependent cytotoxicity function is incorporated herein by reference at this.
As above disclosed, can have people's engineering IL-6 antibody Fc of the present invention district of the effector function of change with design for example by changing C1q combination and/or Fc γ R in conjunction with also therefore having changed CDC activity and/or ADCC activity." effector function " is responsible for activating or weakening biological activity (in the experimenter).The example of effector function includes but not limited to: the C1q combination; Rely on the cytotoxicity (CDC) of complement; The Fc receptors bind; Rely on the cytotoxicity (ADCC) of antibody; Phagolysis; Cell surface receptor is (as B-cell receptor; BCR) following adjusting etc.Such effector function may need the Fc district to combine with land (as antibody variable region) and can use multiple assay method (as Fc in conjunction with measure, ADCC mensuration, CDC mensuration etc.) assess.
For example, can produce the variant Fc district of the Fc γ RIII of C1q combination with improvement and improvement in conjunction with people's engineering IL-6 antibody of (as having ADCC activity and the CDC activity of all improving).Perhaps, if the expectancy effect subfunction reduces or eliminates, but through engineering approaches has the active variant Fc of the ADCC district that the CDC of minimizing is active and/or reduce.In other embodiment, a kind of being increased only arranged, and randomly in these activity, also have other activity to be reduced (have the ADCC activity of improvement as generation, but an active Fc region variants that reduces of CDC, vice versa).
In through engineering approaches, also can import Fc sudden change changing the interaction of they and neonatal Fc receptor (FcRn), and improve their PK (pharmacokinetic) profile.Described already have an improvement with FcRn bonded people Fc variant set (Shields etc., (2001) .High resolutionmapping of the binding site on human IgG1 for Fc γ RI, Fc γ RII, Fc γ RIII, and FcRn and design of IgG1 variants with improved binding to the Fc γ R, J.Biol.Chem.276:6591-6604).
The aminoacid replacement of another kind of type is used to change the glycosylation type in people's engineering IL-6 antibody Fc district.The glycosylation in Fc district is generally N connection or O connection.The N connection refers to that carbohydrate partly is connected on the side chain of asparagine residue.The O linked glycosylation refers to that one of carbohydrate N-acetylgalactosamine, semi-lactosi or wood sugar are connected on the hydroxy-amino-acid, is generally Serine or Threonine most, although also can use 5-oxyproline or 5-oxylysine.Being used for enzyme connection carbohydrate part is l-asparagine-X-Serine and l-asparagine-X-Threonine to the recognition sequence on the l-asparagine side chain peptide sequence, and wherein X is any amino acid except that proline(Pro).Therefore, the existence of any these peptide sequences has just formed the potential glycosylation site in the polypeptide.
One or more glycosylation sites that exist in the polypeptide by disappearance for example, and/or add the swollen non-existent glycosylation site of one or more polypeptide and can change the glycosylation type.Make it to contain one or more above-mentioned tripeptide sequences (being used for N linked glycosylation site) by changing aminoacid sequence, just can realize adding glycosylation site easily to people's engineering IL-6 antibody Fc district.The glycosylation variant of making illustration has the aminoacid replacement of the residue A sn 297 of heavy chain.Also can change by the replacement of original peptide sequence being added one or more Serines or threonine residues or use (being used for O linked glycosylation site).In addition, Asn 297 is changed into Ala and can remove a glycosylation site.
In some embodiments, in the cell of expressing β (1,4)-N-acetylglucosaminyl transferase III (GnTIII), express people's engineering IL-6 antibody of the present invention, make GnT III add GicNAc to people's engineering IL-6 antibody.The method that produces above-mentioned form antibody is provided in WO/9954342, WO/03011878, patent disclosure 20030003097A1 and Umana etc., Nature Biotechnology, 17:176-180, Feb.1999.
Can produce people's anti-IL-6 antibodies randomly by the immune transgenic animal (as mouse, rat, hamster, non-human primate etc.) that can produce as the repertoire of people's antibody said and/or known in the art.The cell that produces people's anti-IL-6 antibodies is separable from such animal, and utilizes the method method for example described herein that is fit to make it immortalization.
Can by currently known methods produce produce and the transgenic mice of the repertoire of human antigen's bonded people antibody (as but be not limited to the U.S. patent No. of Lonberg etc.: 5,770,428,5,569,825,5,545,806,5,625,126,5,625,825,5,633,425,5,661,016 and 5,789,650; WO such as Jakobovits 98/50433, WO such as Jakobovits 98/24893, WO such as Lonberg 98/24884, WO97/13852 such as Lonberg, WO such as Lonberg 94/25585, WO such as Kucherlapate 96/34096, Kucherlapate
Figure A200680024037D0027110330QIETU
Al.EP 0,463 151 B1, EP such as Kucherlapate 0,710 719 A1, the US. patent No.s 5 such as Surani, 545,807, WO such as Bruggemann 90/04036, EP0438 such as Bruggemann 474 B1, EP such as Lonberg 0,814 259 A2, GB such as Lonberg 2 272 440 A, Nature 368:856-859 (1994) such as Lonberg, Taylor etc., Int.Immunol.6 (4) 579-591 (1994), Green etc., Nature Genetics 7:13-21 (1994), Mendez etc., Nature Genetics15:146-156 (1997), Taylor etc., Nucleic Acids Research 20 (23): 6287-6295 (1992), Tuaillon etc., Proc Natl Acad Sci USA 90 (8) 3720-3724 (1993), Lonberg etc., IntRev Immunol 13 (1): 65-93 (1995) and Fishwald etc., and Nat Biotechnol 14 (7): 845-851 (1996), each comfortable this is incorporated herein by reference fully).Generally speaking, these mouse comprise at least a transgenosis that contains from the DNA of at least a human immunoglobulin gene's seat, and this locus is that the functioning gene that maybe can stand that functioning gene is reset is reset.Can destroy or lack endogenous immunoglobulin loci in such mouse to eliminate the ability that animal is produced as the coded antibody of native gene.
Can utilize the peptide display libraries to realize the screening of specificity easily in conjunction with similar protein or segmental antibody.This method comprises the screening for the peptide big collection of the single member with desired function or structure.The antibody screening of peptide display libraries is well-known in the art.The peptide sequence of being showed can be 3-5000 or more a plurality of amino acid longs, is generally 5-100 amino acid long, and often is about 8-25 amino acid long.Except that being used to produce the direct chemical synthesis method of peptide library, some recombinant DNA method have been described.A kind of method is included in displayed polypeptide sequence on phage or the cell surface.Each phage or cell contain the nucleotide sequence of the specific displayed polypeptide sequence of coding.Such method is described in PCT patent publication No. 91/17271,91/18980,91/19818 and 93/08278.
Other systems that are used to produce peptide library have the content of external chemosynthesis and recombination method two aspects.Referring to PCT patent publication No. 92/05258,92/14843 and 96/19256. also referring to the U.S. patent No. 5,658,754; With 5,643,768.Peptide display libraries, carrier and screening reagent box city be sold by such supplier such as Invitrogen (Carlsbad, CA) and Cambridge Antibody Technologies (Cambridgeshire, UK).Referring to as transfer the U.S. patent No. 4704692,4939666,4946778,5260203,5455030,5518889,5534621,5656730,5763733,5767260,5856456 of Enzon; Transfer 5223409,5403484,5571698,5837500 of Dyax; Transfer 5427908,5580717 of Affymax; Transfer 5885793 of Cambridge Antibody Technologies; Transfer 5750373 of Genentech; Transfer 5618920,5595898,5576195,5698435,5693493,5698417 of Xoma; Colligan, the same; Ausubel, the same; Or Sambrook, the same.Also can utilize at least a anti-IL-6 antibodies coding nucleic acid is offered the transgenic animal that produce such antibody in their Ruzhong or Mammals for example goat, ox, horse, sheep, rabbit etc., prepare antibody of the present invention.Can utilize currently known methods that such animal is provided.Referring to such as but not limited to the US patent No. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489 etc., each comfortable this is incorporated herein by reference fully.
Can utilize extraly with at least a anti-IL-6 antibodies coding nucleic acid offer the transgenic plant that produce such antibody and cultivation vegetable cell (as but be not limited to tobacco and corn), come from wherein plant part or specific part or the variant in the cultured cells, prepare antibody of the present invention.As nonrestrictive example, successfully used the transgene tobacco leaf of express recombinant protein that a large amount of recombinant proteins is provided already, as using inducible promoter.Referring to as Cramer etc., Curr.Top.Microbol.Immunol.240:95-118 (1999) and the reference of wherein quoting.Equally, also used transgenic corns to express the mammalian proteins of commercial production levels, have be equivalent in other recombination systems, produce or from those proteic biological activitys of natural origin purifying.Referring to as Hood etc., Adv.Exp.Med.Biol.464:127-147 (1999) and the reference of wherein quoting.Also from the transgenic plant seed that comprises tobacco seed and potato tuber, produce a large amount of antibody, comprised for example single-chain antibody (scFv ' s) of antibody fragment.Referring to as Conrad etc., Plant MoI.Biol.38:101-109 (1998) and the reference of wherein quoting.Therefore, also can utilize transgenic plant to produce antibody of the present invention according to currently known methods.Also referring to as Fischer etc., Biotechnol.Appl.Biochem.30:99-108 (Oct., 1999), Ma etc., Trends Biotechnol.13:522-7 (1995); Ma etc., Plant Physiol.109:341-6 (1995); Whitelam etc., Biochem.Soc.Trans.22:940-944 (1994); And the reference of wherein quoting.
Antibody of the present invention widely avidity (KD) in conjunction with people IL-6.In a preferred embodiment, at least a people mAb of the present invention can be randomly with high-affinity in conjunction with people IL-6.For example, people or people's engineering mAb can be to be equal to or less than about 10 -7The K of M DIn conjunction with people IL-6, such as but not limited to 0.1-9.9 (or wherein any scope or value) * 10 -7, 10 -8, 10 -9, 10 -10, 10 -11, 10 -12, 10 -13, 10 -14, 10 -15Or wherein any scope or value, as by those skilled in the art skilled surperficial plasmon resonance or Kinexa method measured.
Can utilize any suitable method to measure antibody by experiment to antigenic avidity or avidity.(referring to for example, Berzofsky, etc., ＂ Antibody-Antigen Interactions, ＂ be at Fundamental Immunology, Paul, and W.E. compiles, and Raven Press:New York is among the NY (1984); Kuby, Janis Immunology, W.H.Freeman and Company:NewYork, NY (1992); And method described herein).If measure down in different condition (as salt concn, pH), the avidity of specific antibody-AI of being measured can change.Therefore, preferably use antibody and antigenic standardized solution and standardized damping fluid damping fluid for example described here to produce the observed value of avidity and other antigen incorporating parametrics (as K D, K On, K Off).
Available being at war with property of antibody mensuration of the present invention combines IL-6 and/or total epi-position district so that measure what albumen, antibody or other antagonists with antibody competition of the present invention.These are easy to assay method known to those skilled in the art and have assessed between antagonist or the part of competition to(for) the binding site on the albumen of limited quantity.Albumen and/or antibody are fixing or undissolved before or after competition, and will separate with IL-6 bonded sample and unconjugated sample by for example decant (albumen/antibody is not dissolved) or centrifugal (wherein at competing reaction postprecipitation albumen/antibody).Equally, can by function whether by antibody with proteic combine or lack in conjunction with being changed measure competitive combination, whether suppress or strengthen for example enzymic activity of mark as antibody molecule.As known in the art, can use ELISA and other functional assays methods.
Preferred anti-IL-6 antibodies of the present invention has the sequence shown in following table 1-5 and the 12-14.For example, anti-IL-6 antibodies of the present invention has the heavy chain CDR sequence (being CDRH1, CDRH2 and CDRH3) shown in a light chain CDR sequence shown in the table 1 (being CDRL1, CDRL2 and CDRL3) and the table 2.More particularly, anti-IL-6 antibodies (molecule AME-A9) has the CDRH2 of CDRH1, SEQ ID NO:61 of CDRL3, SEQ ID NO:47 of CDRL2, SEQ IDNO:35 of CDRL1, SEQ ID NO:27 of SEQ ID NO:15 and the CDRH3 of SEQ ID NO:91.
Nucleic acid molecule
Use is in this information that provides, for example, coding SEQ ID NOS:93,97 and 101 at least a variable region of light chain, together with other sequences disclosed here, and SEQ IDNOS:95,99 and 103 at least a variable region of heavy chain, together with other sequences disclosed here, their specific fragment, variant or consensus sequence, or comprise the nucleotide sequence of continuous amino acid of 70-100% at least of carrier of the preservation of at least a these sequences, can utilize methods known in the art described here to obtain the nucleic acid molecule of the present invention of at least a anti-IL-6 antibodies of coding.
Nucleic acid molecule of the present invention can rna form exists, and for example mRNA, hnRNA, tRNA or any other form, or exist with dna form include but not limited to by the clone or syntheticly produce the cDNA that obtains and genomic dna or any their combination.DNA can be three chains, two strands or strand, or their any combination.Any part of DNA or RNA at least one chain can be a coding strand, is also referred to as positive-sense strand, or it can be noncoding strand, is also referred to as antisense strand.
Isolated nucleic acid molecule of the present invention can comprise and comprises the open reading-frame (ORF) (ORF) that randomly has one or more introns, as but be not limited to, at least one CDR of at least one heavy chain (as SEQ ID NOS:38,40,42,44 etc.) or light chain (as SEQ ID NOS:2,4,6,8 etc.) is the nucleic acid molecule of the open reading-frame (ORF) (ORF) of at least one specific part of CDR1, CDR2 and/or CDR3 for example; The nucleic acid molecule that comprises anti-IL-6 antibodies or variable region (as SEQ ID NOS:94,98 and 102 variable region of light chain, together with other sequences disclosed here and SEQ ID NOS:96,100 and 104 variable region of heavy chain) encoding sequence; Basically be different from above-mentioned those nucleotide sequence with containing, but because the nucleic acid molecule of the nucleotide sequence of genetic code degeneracy and still encode said and/or at least a anti-IL-6 antibodies known in the art.Certainly, genetic code is well-known in the art.Therefore, to those skilled in the art, the degeneracy nucleic acid variant that produces the specific anti-IL-6 antibodies of the present invention of such coding should be a routine techniques.Referring to as Ausubel, etc., the same, and such nucleic acid variant is included among the present invention.
As shown here, the nucleic acid molecule of the present invention that comprises the nucleic acid of the anti-IL-6 antibodies of encoding can include but not limited to, those nucleic acid molecule of the independent segmental aminoacid sequence of encoding antibody; The complete antibody or the encoding sequence of its part; The encoding sequence of antibody, fragment or part, and extra sequence, the encoding sequence of at least one signal leader sequence or fusogenic peptide for example, have or do not have aforesaid extra encoding sequence, for example at least one intron, and extra non-coding sequence includes but not limited to noncoding 5 ' and 3 ' sequence, for example transcribe, mRNA processing comprises transcribing of working in montage and the polyadenylation signal (for example, rrna combination and the stability of mRNA), the non-transcribed sequence; The extra amino acid of encoding for example provides extra functional those amino acid whose extra encoding sequences.Therefore, the sequence of encoding antibody can be merged to the marker sequence, for example coding help comprises the sequence of the peptide of antibody fragment or fusion antibody purification partly.
Polynucleotide with polynucleotide selective cross described here
The invention provides the isolating nucleic acid of multi-nucleotide hybrid disclosed here under the selective cross condition.Therefore, the polynucleotide of this embodiment can be used for separating, detecting and/or quantitatively comprise the nucleic acid of such polynucleotide.For example, polynucleotide of the present invention can be used for identifying, separate or the preservation library of increasing in part or full-length clone.In certain embodiments, polynucleotide are isolating genome or cDNA sequence, otherwise or with cDNA complementation from people or mammalian nucleic acid library.
Ground preferably, the cDNA library comprises at least 80% full length sequence, preferably, at least 85% or 90% full length sequence, and more preferably, at least 95% full length sequence.But normalized cDNA library increases the representativeness of rare sequence.The hybridization conditions of low or medium strictness normally, but exclusively non-, the sequence that is had the sequence identity of minimizing with respect to complementary sequence is used.In high stringent condition can randomly be used to have the sequence of higher identity.The selective cross of the sequence with about 70% sequence identity is taken place in low stringency condition, and can be used for identifying lineal homology or collateral line homologous sequence.
Randomly, polynucleotide of the present invention should be encoded at least a portion of the antibody of this described polynucleotide encoding of place.Polynucleotide of the present invention comprise the nucleotide sequence that can be used for the polynucleotide selective cross of code book invention antibody.Referring to as Ausubel, the same; Colligan, the same, each comfortable this is incorporated herein by reference fully.
Nucleic acid construct
Can use (a) recombination method as known in the art, (b) synthetic technology, (c) purification technique, and/or (d) their combined preparation isolating nucleic acid of the present invention.
Nucleic acid can comprise the sequence except that polynucleotide of the present invention easily.For example, the multiple clone site that comprises one or more endonuclease restriction sites can be inserted in the nucleic acid to help polynucleotide to separate.Equally, the polynucleotide of the present invention that interpretable sequence can be inserted to help translation separate.For example, 6-histidine mark thing sequence provides easily mode with purifying albumen of the present invention.Nucleic acid of the present invention except that encoding sequence is carrier, linker or the joint for being used for polynucleotide clone of the present invention and/or expressing randomly.
Extra sequence can be added on such clone and/or the expressed sequence to optimize their the clone and/or the function in expressing, help polynucleotide to separate, or improve the polynucleotide transfered cell.The application of cloning vector, expression vector, linker and joint is well-known in the art.(, the same referring to as Ausubel; Or Sambrook, the same).
Be used to make up the recombination method of nucleic acid
Can use multiple cloning process well known by persons skilled in the art to learn and obtain isolating nucleic acid composition of the present invention by biological origin, for example RNA, cDNA, genomic dna or their any combination.In certain embodiments, under stringent condition, be used to the sequence expected in identification of cdna or the genome dna library with the oligonucleotide probe of polynucleotide selective cross of the present invention.The structure of the separation of RNA and cDNA and genomic library is well-known for those skilled in the art.(, the same referring to as Ausubel; Or Sambrook, the same).
Nucleic acid screening and separation method
Can utilize probe screening cDNA or genomic library, for example at these those disclosed polynucleotide sequences based on polynucleotide sequence of the present invention.Probe can be used for genomic dna or cDNA sequence hybridization to separate the homologous gene in the identical or different organism.Those skilled in the art are to be understood that can use the strict degree of different hybridization in this mensuration; And hybridization or washing medium can be strict.When hybridization conditions becomes stricter, between probe that is used to form duplex and target, necessarily there is the complementarity of higher degree to occur.Strict degree can be controlled by for example one or more of existence of methane amide of temperature, ionic strength, pH and partially denaturing solvent.For example, by for example in the 0%-50% scope, handling the polarity that methane amide concentration changes reactant solution, hybridize strict performance and obtain easily changing.Detectable degree in conjunction with needed complementarity (sequence identity) can change along with the severity of hybridization medium and/or washing medium.Complementary degree is preferably 100% or 70-100% or any therein scope or value.Yet, know that very sequence change small in probe and the primer can compensate the hybridization of minimizing and/or the severity of washing medium.
Based on instruction and guidance in this proposition, the method for cloning RNA or DNA is well-known in the art, and can be used according to the present invention and need not undo experimentation.
Known DNA or RNA amplification method include but not limited to polymerase chain reaction (PCR) and relevant amplification method (referring to as Mullis, the U.S. patent No. 4,683,195,4,683,202,4,800,159,4,965,188 that waits; Tabor, wait 4,795,699 and 4,921,794; 5,142,033 of Innis; Wilson, wait 5,122,464; 5,091,310 of Innis; Gyllensten, wait 5,066,584; Gelfand, wait 4,889,818; Silver, wait 4,994,370; 4,766,067 of Biswas; Ringold 4,656,134) and use amplification (Malek at the RNA mediation of the sense-rna that is used for double-stranded DNA synthetic target sequence as template, Deng the U.S. patent No. 5,130,238, have trade(brand)name NASBA), the full text of these documents is incorporated herein by reference at this.(, the same referring to as Ausubel; Or Sambrook, the same).
For example, polymerase chain reaction (PCR) technology can be used for by direct amplification polynucleotide sequence of the present invention of genomic dna or cDNA library and relevant gene.PCR and other amplification in vitro method also can be used for the nucleotide sequence that clones coding is for example treated expressed proteins, and preparation is used as the nucleic acid that probe is used for the existence of the mRNA that test sample expects, is used for nucleic acid sequencing, or is used for other purposes.In amplification in vitro method, be enough to instruct the example of those skilled in the art's technology to see Berger, the same, Sambrook, the same, and Ausubel, the same, and Mullis, etc., the U.S. patent No. 4,683,202 (1987); And Innis, etc., PCRProtocols A Guide to Methods and Applications compiles, Academic PressInc., San Diego, CA (1990).The commercial reagent box that is used for the genome pcr amplification is known in the art.Referring to as Advantage-GC genome PCR test kit (Clontech).In addition, can be used for improving the productive rate of long PCR product as T4 gene 32 albumen (Boehringer Mannheim).
Be used to make up the synthetic method of nucleic acid
Also available known method is by the isolating nucleic acid of direct chemical synthetic preparation the present invention (referring to as Ausubel, etc., the same).Chemosynthesis produces single stranded oligonucleotide usually, and it is by changing double-stranded DNA into complementary sequence hybridization or by utilizing strand to use archaeal dna polymerase to carry out polymerization as template.The sequence of about 100 or more a plurality of bases can be limited to though one of ordinary skill in the art would recognize that the chemosynthesis of DNA, longer sequence can be obtained by connecting shorter sequence.
Recombinant expression cassettes
The present invention further provides the recombinant expression cassettes that comprises nucleic acid of the present invention.Nucleotide sequence of the present invention, for example cDNA of code book invention antibody or genome sequence can be used for making up the recombinant expression cassettes of the host cell that can be imported into a kind of at least expectation.Recombinant expression cassettes comprises operationally usually with transcription initiation regulates the polynucleotide of the present invention that sequence is connected, and described transcription initiation is regulated transcribing of polynucleotide in the host cell of sequence-directed expection.Allos and non-allos (promptly endogenous) promotor all can be used for the direct expression of nucleic acid of the present invention.
In certain embodiments, can will import as promotor, enhanser or the isolating nucleic acid of other elements in the appropriate location of polynucleotide of the present invention of non-allos form (in upstream, downstream or the intron), so that the expression of the polynucleotide of adjusted or the invention of downward modulation abridged edition.For example, can and/or replace in the body or external change endogenesis promoter by sudden change, disappearance.
Carrier and host cell
The invention still further relates to the carrier that comprises isolated nucleic acid molecule of the present invention, use the genetically engineered host cell of this recombinant vectors, and produce at least a anti-IL-6 antibodies by recombinant technology well-known in the art.Referring to as Sambrook, etc., the same; Ausubel, etc., the same, each comfortable this is incorporated herein by reference fully.
Polynucleotide can randomly be connected to and be used in the carrier that contains selective marker that the host breeds.Generally speaking, for example with the form of calcium phosphate precipitation, or import plasmid vector with precipitation with the form that forms mixture with charged lipids.If carrier is a virus, then can utilize suitable packing cell to tie up to external packing, transduction is gone in the host cell then.
DNA inserts fragment and should be operationally be connected with suitable promotor.Expression construct further contains and is useful on transcription initiation, terminated site, and in transcriptional domain, contains the ribosome bind site that is useful on translation.The terminator codon (as UAA, UGA or UAG) that the encoding part of the mature transcript of expressing by construct preferably includes the transcription initiation codon that is positioned at beginning and suitably is positioned over mRNA end to be translated is for Mammals or preferred UAA of eukaryotic cell expression and UAG.
Expression vector preferably but randomly comprise at least a selective marker.Such mark comprises, as but be not limited to for eukaryotic cell culture methotrexate (MTX), Tetrahydrofolate dehydrogenase (DHFR, the US patent No. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017), penbritin, Xin Meisu (G418), mycophenolic acid or glutamine synthase (GS, the US patent No. 5,122,464; 5,770,359; 5,827,739) resistant gene, and in intestinal bacteria and other bacteriums or prokaryotic organism, cultivating tsiklomitsin or ampicillin resistance gene (above-mentioned patent is incorporated herein by reference fully at this).The suitable substratum and the culture condition that are used for above-mentioned host cell are known in the art.The carrier that is fit to is conspicuous for those skilled in the art.Can pass through the transfection that calcium phosphate transfection, DEAE-dextran be situated between, transfection, electroporation, transduction, infection or other known methods of cation lipid mediation imports the vector construction body in the host cell.Such method is described and is described in the art, and for example Sambrook is the same, 1-4 and 16-18 chapter; Ausubel, the same, the 1st, 9,13,15,16 chapters.
At least a antibody of the present invention can be expressed by the modification type, fusion rotein for example, and not only can comprise secretion signal, and can comprise extra allos functional zone.For example, can be with extra amino acid district, particularly charged amino acid district, the N-end that adds antibody to is to improve in purifying or subsequent operations and stability and the persistence of duration of storage in host cell.Equally, peptide moiety can be added on the antibody of the present invention and be beneficial to purifying.Can before antibody or the final preparation of its at least one fragment, remove such zone.Such method is described in many standard laboratory handbooks, and for example Sambrook is the same, 17.29-17.42 and 18.1-18.74 chapter; Ausubel, the same, the 16th, 17 and 18 chapters.
Those skilled in the art can be used to express code book and invent proteic expression of nucleic acids system and have at fingertips for multiple.Perhaps, can in host cell, express nucleic acid of the present invention by opening the host cell that (by handling) contains the interior source DNA of code book invention antibody.Such method is well-known in the art, as is described in the US patent No. 5,580,734,5, in 641,670,5,733,746 and 5,733,761, is incorporated herein by reference fully at this.
The illustrative cell culture that is used to produce antibody, its specific part or variant is a mammalian cell.The all normal forms with monolayer cell of mammal cell line exist, although can also use mammalian cell suspension or bio-reactor.Developed the host cell that the is fit to system of the glycosylated protein of multiple energy The expressed in the art already, comprise COS-1 (as ATCC CRL 1650), COS-7 (as ATCC CRL-1651), HEK293, BHK21 (as ATCC CRL-10), CHO (as ATCC CRL 1610) and BSC-1 (as ATCC CRL-26) clone, the Cos-7 cell, Chinese hamster ovary celI, hep G2 cell, P3X63Ag8.653, SP2/0-Ag14,293 cells, HeLa cell etc., certainly for example can easily obtain, American type culture collection, Manassas, Va (www.atcc.org).Preferred host cell comprises the cell in lymph source, for example myelomatosis and lymphoma cell.Particularly preferred host cell is P3X63Ag8.653 cell (ATCC preserving number CRL-1580) and SP2/0-Ag14 cell (ATCC preserving number RL-1851).In an especially preferred embodiment, reconstitution cell is P3X63Ab8.653 or SP2/0-Ag14 cell.
The expression vector that is used for these cells can comprise one or more following expression control sequencs, such as but not limited to, replication orgin; Promotor is (as late period or early stage SV40 promotor, CMV promotor (the US patent No. 5,168,062; 5,385,839), HSV tk promotor, pgk (phosphoglyceric kinase) promotor, EF-1 α promotor (the US patent No. 5,266,491), at least a humen immunoglobulin storter); Enhanser and/or machining information site, for example ribosome bind site, RNA splice site, polyadenylation site (adding the site) and transcription termination sequence as the big T Ag of SV40 polyadenylic acid.Referring to as Ausubel etc., the same; Sambrook, etc., the same.Other are used to produce nucleic acid of the present invention or proteic cell is known and/or can obtains from for example American type culture collection clone and hybridoma catalogue (www.atcc.org) or other known commercial source.
When using eukaryotic host cell, usually polyadenylation or transcription termination sequence are mixed in the carrier.An example of terminator sequence is the polyadenylation sequence from bovine growth hormone gene.Also can comprise the sequence that is used for the correct montage of transcript.An example of montage sequence is the VP1 intron (Sprague, etc., J.Virol.45:773-781 (1983)) from SV40.In addition, as known in the art, the gene order that regulation and control in the host cell are duplicated can be mixed in the carrier.
Antibody purification
Can from the reconstitution cell culture, reclaim and the purifying anti-IL-6 antibodies by well-known method, include but not limited to albumin A purifying, ammonium sulfate or ethanol sedimentation, sour extracting, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography.High performance liquid chromatography (" HPLC ") also can be used for purifying.Referring to as Colligan, Current Protocols in Immunology, or CurrentProtocols in Protein Science, John Wiley ﹠amp; Sons, NY, NY, (1997-2001), as the 1st, 4,6,8,9,10 chapters, each comfortable this is incorporated herein by reference fully.
Antibody of the present invention comprise natural purifying product, chemical synthesis process product and by the product that comprises that the eucaryon host of yeast, higher plant, insect and mammalian cell for example produces by recombinant technology.Depend on employed host in the recombinant production process, antibody of the present invention can be glycosylated or nonglycosylated, and is preferably glycosylated.Such method is described in many standard laboratory handbooks, and for example Sambrook is the same, the 17.37-17.42 joint; Ausubel, the same, the 10th, 12,13,16,18 and 20 chapters and sections; Colligan, ProteinScience, the same, the 12-14 chapter, each comfortable this is incorporated herein by reference fully.
Anti-IL-6 antibodies
Anti-IL-6 antibodies according to the present invention comprises any albumen or peptide that contains molecule, described molecule comprises at least a portion immunoglobulin molecules, such as but not limited at least one ligand binding moiety (LBP), complementarity-determining region (CDR) or its ligand binding moiety such as but not limited to heavy chain or light chain, heavy chain or variable region of light chain, framework region is (as FR1, FR2, FR3, FR4 or its fragment, or shown in SEQ ID NOS:105-112, further randomly comprise at least one replacement, insert or disappearance), heavy chain or constant region of light chain (as comprise at least one CH1, hinge area 1 (hingel), hinge area 2, hinge area 3, hinge area 4, CH2 or CH3 or its fragment further randomly comprise at least one replacement, insert or disappearance) or its any part that can incorporate in the antibody of the present invention.Antibody of the present invention can comprise maybe can derive from any Mammals, such as but not limited to people, mouse, rabbit, rat, rodent, primate or their any combination etc.
Isolated antibody of the present invention is included as any suitable coded antibody aminoacid sequence disclosed here of polynucleotide, or any isolating or the preparation antibody.Ground preferably, people's antibody or Fab are in conjunction with people IL-6 and so the part or the proteic at least a biological activity that neutralizes basically.Part or preferably neutralize basically at least a IL-6 albumen or segmental at least a bioactive antibody or its specific part or variant can be in conjunction with this albumen or fragments, and therefore suppress the activity that mechanism mediated that rely on or that mediate by IL-6 and IL-6 receptors bind or other IL-6.Term " neutralizing antibody " refers to depend on measuring method as used herein, suppress about 20-120%, preferably at least about 10,20,30,40,50,55,60,65,70,75,80,85,90,91,92,93,94,95,96,97,98,99,100% or the active antibody that relies on of more IL-6.Preferably suppress the active ability that IL-6 relies on by at least a as said and/or known in the art suitable IL-6 albumen or receptor determination method assessment anti-IL-6 antibodies.People's antibody of the present invention can be any kind (IgG, IgA, IgM, IgE, IgD etc.) or isotype, and can comprise κ or lambda light chain.In one embodiment, people's antibody comprises IgG heavy chain or regulation fragment, for example at least a isotype, IgG1, IgG2, IgG3 or IgG4 (as γ 1, γ 2, γ 3 or γ 4).Can comprise the antibody that at least a as people's light chain said and/or known in the art (as IgG, IgA and IgM) genetically modified transgenic mice or other transgenic nonhuman mammals prepare the type by use.In another embodiment, anti-people IL-6 people antibody comprises IgG1 heavy chain and IgG1 light chain.
At least a defined epitope that is specific at least one IL-6 albumen, its subunit, fragment, part or any combination of at least a antibodies of the present invention.At least a epi-position can comprise at least a antibodies district that comprises albumen at least a portion, and this epi-position preferably partly is made up of outside at least a proteic born of the same parents, soluble, hydrophilic, surperficial or tenuigenin.Complete specific part for the continuous amino acid of SEQ ID NO:115, any combination that at least a defined epitope can comprise 1-3 at least amino acid whose at least a aminoacid sequence for example, amino-acid residue 151-178 position, more particularly, residue 171-182 position.
Generally speaking, people's antibody of the present invention or Fab should comprise at least one individual complementarity-determining region (CDR1, CDR2 and CDR3) or the variant that contains at least one variable region of heavy chain, and the antigen binding domain of at least one individual complementarity-determining region of at least one variable region of light chain (CDR1, CDR2 and CDR3) or variant.The CDR sequence can derive from ethnic group be sequence or with kind be that sequence matches closely.For example, can use CDRs from the synthetic library that derives from original mouse CDRs.Can mix original mouse sequence and form these CDRs by guarding to replace.As nonrestrictive example, antibody or antigen-binding portion thereof or variant can comprise at least one to have the heavy chain CDR3 that is selected from SEQ ID NOS:79,81,83,85,87,89 and 91 aminoacid sequence and/or has the light chain CDR3 that is selected from SEQ ID NOS:29,31,33 and 35 aminoacid sequence.In a specific embodiment, antibody or Fab can have the antigen binding domain of at least a portion that comprises at least one heavy chain CDR (being CDR1, CDR2 and/or CDR3), and described at least one heavy chain CDR has the aminoacid sequence (as SEQ ID NOS:37,49 and 79) of corresponding C DRs 1,2 and/or 3.In another specific embodiment, antibody or antigen-binding portion thereof or variant can have the antigen binding domain of at least a portion that comprises at least one light chain CDR (being CDR1, CDR2 and/or CDR3), and described at least one light chain CDR has the aminoacid sequence (as SEQ ID NOS:1,17 and 29) of corresponding C DRs 1,2 and/or 3.
In a preferred embodiment, 3 of antibody or Fab heavy chain CDRs and 3 light chain CDRs have the aminoacid sequence of the corresponding C DR of at least a mAb AME-A9 as described herein, AME-1b, AME-18a, AME-22a, AME-20b, AME-23a and AME-19a.Can by use ordinary method with different piece (as CDRs, the framework region) chemistry of antibody link together, by the ordinary method preparation of using recombinant DNA technology and (promptly one or more) nucleic acid molecule of expressing this antibody of coding or by using the antibody of any other suitable method preparing.
Anti-IL-6 antibodies can comprise the heavy chain or the variable region of light chain of at least one aminoacid sequence with regulation.For example, in a preferred embodiment, anti-IL-6 antibodies comprise at least one at least one randomly have the variable region of heavy chain that is selected from SEQ ID NOS:95,99,103,118,122,126 and 130 aminoacid sequence, and/or at least one randomly has the variable region of light chain that is selected from SEQ ID NOS:93,97,101,116,120,124 and 128 aminoacid sequence.Can use the preparation of suitable method in conjunction with people IL-6 and comprise the heavy chain of regulation or the antibody of variable region of light chain, phage display (Katsube for example, Y., Deng, Int J Mol.Med, 1 (5): methods 863-868 (1998)) or as known in the art and/or use transgenic animal described here.For example, can choose IL-6 or its fragment immunity comprise function resets human immunoglobulin heavy chain's transgenosis and contains to come self energy to stand the genetically modified transgenic mice of the DNA of the human normal immunoglobulin light chain gene seat that function resets, produces to excite antibody.Optionally, can produce cell and prepare hybridoma or the antibody produced cell of other immortalizations by separation antibody as described herein and/or known in the art.Perhaps, can in the host cell that is fit to, use coding nucleic acid or its part expressing antibodies, specific part or variant.
The amino acid coding
Form that the amino acid of anti-IL-6 antibodies of the present invention normally abridges.Can indicate amino acid by its single-letter coding well-known in the art, its trigram coding, title or trinucleotide codon, indicate the amino acid title (referring to Alberts, B., Deng, Molecular Biologyof The Cell, the 3rd edition., Garland Publishing, Inc., New York, 1994).
Anti-IL-6 antibodies of the present invention can comprise one or more as be cited from spontaneous mutation or artificial aminoacid replacement, disappearance or the interpolation of handling at this.Can identify in the anti-IL-6 antibodies of the present invention for the requisite amino acid of function by methods known in the art, and for example site-directed mutagenesis or alanine scanning mutagenesis (as Ausubel, the same, the 8th, 15 chapter; Cunningham and Wells, Science 244:1081-1085 (1989)).A kind of method in back imports to single alanine mutation at each residue place of molecule.Test the biological activity of resulting mutating molecule then, such as but not limited among at least a IL-6 and active.Also can identify the antibodies critical sites by structural analysis, for example crystallization, nucleus magnetic resonance or photoaffinity labeling (Smith, etc., J.MoI.Biol.224:899-904 (1992) and de Vos, etc., Science 255:306-312 (1992)).
Anti-IL-6 antibodies of the present invention can include but not limited to SEQ ID NOS:91,93,95,97,99 etc. one of at least be selected from 5 at least a portion, sequence or combination to all continuous amino acids.
Energy increases or keeps at least a listed active non-limiting variant to include but not limited to any aforementioned polypeptides, further comprises at least one sudden change corresponding to the replacement at least one residue that changes between disclosed variant aminoacid sequence.
Polypeptide with the aminoacid sequence that is different from continuous amino acid sequence one of at least such as SEQ ID NOS:95,99 and 103 etc. (as the one or more conservative replacement of the sequence that provides at this) can further randomly be provided anti-IL-6 antibodies.Equally, the present invention comprises the variant of SEQ ID NOS:93,97 or 101 light chain variable region amino acid sequence or SEQ ID NOS:79,81,83,85,87,89 or 91 heavy chain amino acid sequence.
As skilled in the art to understand, the present invention includes at least a biologically active antibody of the present invention.Biologically active antibody has natural (nonsynthetic), endogenous or relevant and at least 20%, 30% or 40% of known antibodies, and preferably at least 50%, 60% or 70%, and most preferably at least 80%, 90% or 95%-1000% or more specific activity.The assay method of enzymic activity and substrate specificity and quantitative method are that those skilled in the art are well-known.
On the other hand, the present invention relates to described herein by covalently bound organism part adorned people's antibody and Fab.Such modification can produce the antibody or the Fab of the PK (pharmacokinetic) profile (as serum half-life in the body that increases) with improvement.The organism part can be line style or side chain hydrophilic polymer group, fatty acid group or fatty acid ester group.In specific embodiment, the hydrophilic polymer group can have about 800-about 120,000 daltonian molecular weight, and can be polyalkane glycol (as polyoxyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, aminoacid polymers or polyvinylpyrrolidone, and lipid acid or fatty acid ester group can comprise about 40 carbon atoms of about 8-.
Antibody that the present invention modifies and Fab can comprise one or more and the organism part direct or indirect covalent bonding of antibody.The organism part of each and antibody of the present invention or Fab bonding can be hydrophilic polymer group, fatty acid group or fatty acid ester group independently.Term " fatty acid " as used herein " comprise monocarboxylic acid and dicarboxylic acid." hydrophilic polymer group " refers to compare with octane organic polymer more soluble in water during as term as used herein.For example, to compare polylysine more soluble in water with octane.Therefore, the antibody of modifying by covalently bound polylysine is contained by the present invention.The hydrophilic polymer that is suitable for modifying antibody of the present invention can be line style or side chain, comprises for example polyalkols (as PEG, methoxy poly (ethylene glycol) (mPEG), PPG etc.), carbohydrate (as dextran, Mierocrystalline cellulose, oligosaccharides, polysaccharide etc.), hydrophile amino acid polymer (as polylysine, poly arginine, poly aspartic acid etc.), polyalkane oxide compound (as polyethylene oxide, poly(propylene oxide) etc.) and polyvinyl arsenic pyrrolidone.Ground preferably, the hydrophilic polymer of modifying antibody of the present invention has about 800-about 150,000 daltonian molecular weight as independent molecular entity.For example, can use the PEG that wherein is designated as the polymkeric substance molecular-weight average of representing with dalton down 5000And PEG 20,000The hydrophilic polymeric group can replace with about 6 alkyl of 1-, lipid acid or fatty acid ester group.Can be by the hydrophilic polymer that uses the appropriate means preparation to replace with lipid acid or fatty acid ester group.For example, can will comprise polymkeric substance and the fatty acid carboxylate ester or the fatty acid ester coupling of amido, then on the lipid acid activatory carboxylicesters (as using N, N-carbonyl dimidazoles activatory) or fatty acid ester can with the hydroxyl coupling on the polymkeric substance.
Be suitable for modifying the lipid acid of antibody of the present invention and fatty acid ester and can be and saturated maybe can contain one or more unsaturated units.The lipid acid that is suitable for modifying antibody of the present invention comprises, for example n-dodecane hydrochlorate (C 12, lauroleate), n-tetradecane hydrochlorate (C 14, myristate), Octadecane hydrochlorate (C 18, stearate), NSC 62789 hydrochlorate (C 20, the peanut hydrochlorate), n-docosane hydrochlorate (C 22, the mountain hydrochlorate), positive melissate (C 30), positive tetracontane hydrochlorate (C 40), suitable-Δ 9-octadecane hydrochlorate (C 18, oleate), all suitable-Δ 5,8,11,14-Eicosatetraenoic hydrochlorate (C 20, arachidonate), suberic acid, tetradecane diacid, octadecane diacid, docosandioic acid etc.The fatty acid ester that is fit to comprises the monoesters of the dicarboxylic acid that comprises line style or side chain low alkyl group.Low alkyl group can comprise about 12 of 1-, preferably about 6 carbon atoms of 1-.
The people's antibody and the Fab that can use the appropriate means preparation to modify are for example by reacting in one or more properties-correcting agent." properties-correcting agent " refers to comprise the suitable organic group (as hydrophilic polymer, lipid acid or fatty acid ester) of activating group during as term as used herein." activating group " be under suitable condition can and the reaction of another chemical group between properties-correcting agent and another chemical group, form the chemical part or the functional group of covalent linkage thus.For example, the reactive activating group of amine comprises electrophilic group, for example tosylate, methanesulfonates, halogen (chlorine, bromine, fluorine, iodine), N-hydroxy-succinamide ester (NHS) etc.Can comprise for example maleimide, iodoacetyl (iodoacetyl), acryl (acrylolyl), pyridyl disulfide, 5-sulfydryl-2-nitrobenzoic acid mercaptan (TNB-mercaptan) etc. with the activating group of thiol reactant.Can be with aldehyde functional group and the molecule coupling that contains amine or hydrazides, azido-can form phosphoramidate or phosphinylidyne imines (phosphorimide) bonding with the three valent phosphors radical reaction.Being suitable for activating group the method in the molecule of importing is (referring to for example, Hermanson, G.T., BioconjugateTechniques, Academic Press:San Diego, CA (1996)) known in the art.But activating group Direct Bonding organic group (as hydrophilic polymer, lipid acid, fatty acid ester), or by the shank combination, for example divalence C 1-C 12Group, the heteroatoms that wherein one or more carbon atoms can be such as oxygen, nitrogen or sulphur replaces.The shank that is fit to comprises, for example, Tetraglycol 99 ,-(CH 2) 3-,-NH-(CH 2) 6-NH-,-(CH 2) 2-NH-and-CH 2-O-CH 2-CH 2-O-CH 2-CH 2-O-CH-NH-.Can be for example by in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), allow list-Boc-alkyl diamine (as list-Boc-quadrol, list-Boc-hexanediamine) and fatty acid response being formed on the amine key between unhindered amina and the fatty acid carboxylate ester, thereby produce the properties-correcting agent that comprises shank.Can remove the Boc blocking group from product by handling with trifluoroacetic acid (TFA); as described with expose can with another carbonic ether link coupled primary amine, maybe can allow Boc blocking group and maleic anhydride reaction and make the product cyclisation that obtains with generation activatory lipid acid maleimide derivatives.(referring to for example, Thompson, etc., WO 92/16221, and it is complete to be taught in this and to be incorporated herein by reference).
Can allow the reaction of people's antibody or Fab and properties-correcting agent produce the antibody of modification of the present invention.For example, by using for example NHS ester of PEG of amine active modifier, the organism part can non-locus specificity mode bonding antibody.Also can prepare people's antibody or the Fab of modifying by the disulfide linkage (as intrachain disulfide bond) of going back original antibody or Fab.Then, allow the reaction of institute's reductive antibody or Fab and mercaptan active modifier to produce the antibody of modification of the present invention.Can use the appropriate means preparation to comprise the people's antibody and the Fab of the organism modification partly in bonding antibodies specific of the present invention site, for example reverse proteolysis (reverse proteolysis) (the Fisch et.al. of described method, Bioconjugate Chem., 3:147-153 (1992); Werlen et.al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et.al., Protein Sci 6 (10): 2233-2241 (1997); Itoh et.al., Bioorg.Chem., 24 (1): 59-68 (1996); Capellas et.al., Biotechnol.Bioeng., 56 (4): 456-463 (1997)) and at Hermanson, the method described in the G.T., BioconjugateTechniques, Academic Press:San Diego, CA (1996).
Antiidiotypic antibody composition at anti-IL-6 antibodies
Except that the mono-clonal anti-IL-6 antibodies, it is (anti--Id) antibody to the invention still further relates to the antiidiotype that is specific to such antibody of the present invention.Anti--Id antibody is a kind of antibody of discerning relevant with the antigen binding domain of another antibody usually unique determinant.Zone immunity that can be by containing CDR with antibody or its resists-Id as animal (as the mouse species) preparation of the mutually of the same race and gene type of Id antibody sources.The animal of immunity can discern and respond to the idiotypic determinant of immune antibody and produce anti--Id antibody.Anti--Id antibody also can be used as " immunogen " and induce immunne response in another animal, produces so-called anti--Id antibody.
The present invention also provides at least a anti-IL-6 antibodies composition, comprise therein as at least a, at least two kinds, at least three kinds, at least four kinds, at least five kinds, at least six kinds or more kinds of anti-IL-6 antibodies said and/or known in the art, the form of the composition that described antibody exists with non-natural, mixture or type provides.Such composition comprises the composition that non-natural exists, and the composition that described non-natural exists contains variant, structural domain, fragment or the specific variants of total length, C-and/or N-terminal deletion of anti-IL-6 antibodies aminoacid sequence of continuous amino acid of the 70-100% of at least a or two SEQ of being selected from IDNOS:1-114 and 116-138 or its specific fragment, structural domain or variant.Preferred anti-IL-6 antibodies composition comprises for example SEQ ID NOS:15,27,35,47,61 and 91 or at least a CDR of conduct of its specific fragment, structural domain or variant or at least a or two total lengths, fragment, structural domain or the variant that LBP comprises part of 70-100% of anti-IL-6 antibodies sequence described herein.Further preferred compositions comprises, for example, and the SEQ ID NOS:93 of at least a 70-100% of 40-99%, 95,97,99,101,103 etc. or its specific fragment, structural domain or variant.The percentage ratio of such composition be as known in the art or weight described here, volume, concentration, molconcentration percentage ratio or liquid or drying solution, mixture, suspension, milk sap, particle, powder or colloidal molconcentration.
The antibody compositions that comprises extra therapeutic activity composition
Antibody compositions of the present invention can randomly further comprise at least a compound or the albumen of significant quantity, and described compound or albumen are selected from least a anti-infective, cardiovascular (CV) system medicine, central nervous system (CNS) medicine, autonomous nervous system (ANS) medicine, respiratory tract medicine, stomach and intestine (GI) road medicine, hormone drug, the medicine that is used for water or electrolyte balance, hematologic drug, antitumor agent, immunomodulator, medicament for the eyes, ear medicine or nose medicine, topical drug, nutritional drugs etc.Such medicine is well-known in the art, and composition, indication, dosage and the drug regimen that comprises every kind of medicine all listed in this (referring to as Nursing 2001 Handbook of Drugs, the 21st edition, Springhouse Corp., Springhouse, PA, 2001; HealthProfessional ' s Drug Guide 2001, editor, Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ; Pharmcotherapy Handbook, Wells etc. compile Appleton ﹠amp; Lange, Stamford, CT, each comfortable this is incorporated herein by reference fully).
Anti-infective can be selected from amebicide or at least a antiprotozoal drug, vermifuge, antifungal drug, antimalarial drug, antitubercular agent or at least a antileprotic, glucosamine glycoside medicine, penicillin medicine, cephalosporins medicine, tetracycline medication, sulfa drugs, chlorine quinolones, antiviral drug, macrolide anti-infective and miscellany anti-infective at least a.Cardiovascular drug can be selected from short cardiac inotropic drug, antiarrhythmics, anti-anginal drug, antihypertensive drug, antilipemic and miscellany cardiovascular drug at least a.The CNS medicine can be to be selected from least a of non-narcotic analgesics or to be selected from least a of febrifuge, nonsteroidal anti-inflammatory agent, narcotic or at least a opium anodyne, sedative hypnotic, anticonvulsive agent, thymoleptic, anxiolytic, antipsychotic drug, central nervous system stimulant, antiparkinsonian drug and miscellany central nervous system medicine.The ANS medicine can be selected from cholinergic agent (parasympathomimetic agent), anticholinergic, adrenergic agent (sympathomimetic), adrenergic blocking drug (sympathicolytic), skeletal muscular relaxant and neuromuscular blocking agents at least a.The respiratory tract medicine can be selected from antihistaminic, bronchodilator, expectorant or at least a cough medicine and miscellany respiratory system drug at least a.Stomach and intestine (GI) road medicine can be selected from antacid or at least a sorbent material or at least a antiflatulent, digestive ferment or at least a gallbladdergallstonecholetithiasis solubilizing agent, diarrhea, laxative, antiemetic and anti-ulcerative drug at least a.Hormone drug can be selected from cortin, male sex hormone or at least a anabolic steroid, oestrogenic hormon or at least a progestin, chorionic gonadotrophin, antidiabetic drug or at least a glucagon, Triiodothyronine, Triiodothyronine antagonist, pituitrin and parathyroid gland sample medicine at least a.The medicine that is used for fluid and electrolyte balance can be selected from diuretic(s), ionogen or at least a substitutional solution, souring agent or at least a basifier at least a.Hematologic drug can be selected from hematopoietic, anti-coagulant, blood derivatives and thrombolytic enzymes at least a.Antitumor agent can be selected from alkanisation medicine, metabolic antagonist, microbiotic antitumor agent, the antitumor agent that changes hormonal equilibrium and miscellany antitumor agent at least a.Immunomodulator can be selected from immunosuppressor, vaccine or at least a toxoid, toxinicide or at least a antivenin, immune serum and biological answer-reply instrumentality at least a.Medicament for the eyes, ear medicine and nose medicine can be that to be selected from eye at least a with vasoconstrictor, miscellany medicament for the eyes, ear medicine and nose medicine with antiphlogiston, myotic, iridodilator, eye with anti-infective, eye.Topical drug is selected from local at least a of anti-infective, Scabicide or at least a pediculicide or topical corticosteroid of using.Nutritional drugs can be selected from VITAMIN, mineral substance or heat material (calorics) at least a.Referring to content, the same as Nursing 2001 Drug Handbook.
At least a amebicide or antiprotozoal drug can be selected from atovaquone, chloroquine hydrochloride, chloroquini phosphas, metronidazole, hydrochloric acid metronidazole and Pentamidine Isethionate at least a.At least a vermifuge can be selected from Vermox, pamoic acid quinoline-pyrimidine and Top Form Wormer at least a.At least a antifungal drug can be selected from amphotericin B, amphotericin B cholesterol sulfuric ester mixture, AM Bison mixture, AM Bison, fluconazole, flucytosine, griseofulvin microsize, griseofulvin ultramicrosize, itraconazole, KETOKONAZOL, nysfungin and Terbinafine Hcl at least a.At least a antimalarial drug can be selected from chloroquine hydrochloride, chloroquini phosphas, doxycycline, hydroxychloroquine sulfate, Mefloquine Hydrochloride, PRIMAQUINE PHOSPHATE, Pyrimethamine hcl and Pyrimethamine-Sulfadoxine at least a.At least a antitubercular agent or antileprotic can be selected from Rimonophenazine, seromycin, dapsone, ebutol, vazadrine, pyrazinoic acid amide, Mycobutin, Rifampin, rifapentine and Vetstrep at least a.At least a aminoglycoside medicaments can be selected from amikacin sulfate, Gentamicin Sulfate, neomycinsulphate, Vetstrep and Tobramycin Sulphate at least a.At least a penicillin medicine can be to be selected from amoxicillin/clavulanate potassium, amoxicilin trihydrate, penbritin, sodium ampicillin, three water penicillin Gs, sodium ampicillin/Sulbactam Sodium, cloxacillin sodium, Stampen, Sodium mezlocillin, Nafcillin sodium, oxacillin sodium, benzathine penicillin G, penicilline g potassium, neoproc, Benzylpenicillin sodium, potassium v calcium, piperacillin sodium, piperacillin sodium/tazobactam sodium, ticarcillin disodium and ticarcillin disodium/Clavulanic Potassium at least a.At least a cephalosporins medicine can be to be selected from cefaclor, S 578, the cefonicid sodium element, Cefdinir, the hydrochloric acid Cefepime, Cefixime Micronized, cefmetazole sodium, cefonicid sodium, T-1551, cefotaxime sodium, Cefotetan Disodium, cefoxitin sodium, cefoxitin sodium, cefprozil, ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium, Zinnat, Cefuroxime Sodium, Cefuroxime Sodium, one water Sporidex, cefradine and Loracarbef at least a.At least a tetracycline medication can be selected from demethylchlortetracyclini chloridum, doxycycline calcium, abadox, Doxycycline Hyclate, doxycycline monohydrate, Minocycline Hydrochloride and tetracycline hydrochloride at least a.At least a sulfa drugs can be selected from sulfamethoxazole, Sulphadiazine Sodium, sulfamethoxazole, Sulfafurazole and Sodium Sulfacetamide isoxazole at least a.At least a chlorine quinolones can be that to be selected from Alatrofloxacin, Ciprofloxacin, enoxacin, levofloxacin, lomefloxacin hydrochloride, Nalidixic Acid, Norxin, Ofloxacine USP 23, AT-4140 and trovafloxacin mesylate at least a.At least a fluoroquinolones can be selected from my Qu Sha, Ciprofloxacin star, enoxacin, levofloxacin, lomefloxacin hydrochloride, Nalidixic Acid, Norxin, Ofloxacine USP 23, AT-4140 and trovafloxacin mesylate at least a.At least a antiviral drug can be to be selected from abacavir sulfate, Sodium acyclovir, Amantadine Hydrochloride, amprenavir, cidofovir, delavirdine mesylate, Didanosine, efavirenz, famciclovir, Fomivirsen sodium, Trisodium phosphonoformate hexahydrate, ganciclovir, indinavir sulfate, lamivudine, lamivudine/zidovudine, Viracept, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, Saquinavir, Ro-31-8959, stavudine, valaciclovir hydrochlordide, zalcitabine, zanamivir and zidovudine at least a.At least a macrolide anti-infective can be selected from azithromycin, Clarithromycin, dirithromycin, Abboticine alkali, Stellamicina, erythromycin ethylsuccinate, Erythromycin Lactobionate and Meberyt at least a.At least a miscellany anti-infective can be selected from aztreonam, bacitracin, chloramghenicol sodium succinate, Dalacina, clindamycin palmitate hydrochloride, cleocin phosphate, imipenum sodium and cilastatin sodium, times energy, nitrofurantoin macrocrystals, crystallite furadantin, Quinupristin/dalfopristin, Spectinomycin HCL (Veterinary), Spectinomycin HCL (Veterinary) and vancomycin hydrochloride at least a.(referring to 24-214 page or leaf) as Nursing 2001 Drug Handbook.
At least a short cardiac inotropic drug can be selected from amrinone lactate, digoxin and milrinone lactate at least a.At least a antiarrhythmics can be to be selected from vidarabine, L-3428, Tropintran, bretylium tosylate, diltiazem hydrochloride, Dimpyramidum, disopyramide phosphate, esmolol hydrochloride, flecainide acetate, the fumaric acid ibutilide, Xylotox, mexiletine hydrochloride, Moracizine Hydrochloride, Phenytoin Sodium Salt, phenytoin Sodium, procainamide hydrochloride, propafenone hydrochloride, propranolol hydrochloride, Quinidine Bisulfate, quinaglute, galactoquin, quinidine sulfate, sotalol, Tocainide Hydrochloride and verapamil hydrochloride at least a.At least a anti-anginal drug can be selected from amlodipine besylate, n-Pentyl nitrite, Bepridil hydrochloride, diltiazem hydrochloride, sorbide nitrate, elanfan long, nadolol, nicardipine hydrochloride, nifedipine, nitroglycerine, propranolol hydrochloride, verapamil and verapamil hydrochloride at least a.At least a antihypertensive drug can be to be selected from Acebutolol, Amlodipine Besylate, atenolol USP 23, benazepril hydrochloride, betaxolol hydrochloride, the bisoprolol fumarate, candesartan cilexetil, captopril, carteolol hydrochloride, carvedilol, clonidine, Clonidine Hydrochloride, diazoxide, diltiazem hydrochloride, doxazosin mesylate, enalaprilat, grace naphthalene Puli, eprosartan mesilate, felodipine, the methylsulfonic acid Fenoldopam, fosinopril sodium, the acetic acid guanabenz, guanadrel sulfate, hydrochloric acid paddy ammonia process is new, hydralazine hydrochloride, irbesartan, Isradipine, labetalol hydrochloride, lisinopril, LOSARTAN POTASSIUM, methyldopa, methyldopate hydrochloride, the succsinic acid metoprolol, Metoprolol Tartrate, minoxidil, moexipril hydrochloride, nadolol, nicardipine hydrochloride, nifedipine, nisoldipine, Sodium Nitroprusside, penbutolol sulfate, the Perindopril tert-butyl amine, phentolamine mesilate, pindolol, PRAZOSINI HYDROCHLORIDE, propranolol hydrochloride, quinapril hydrochloride, Ramipril, telmisartan, Vasocard, timolol maleate, Trolapril, valsartan and verapamil hydrochloride at least a.At least a antilipemic can be selected from atorvastatincalcuim, cerivastatin sodium, QUESTRAN, Colestipol Hydrochloride, fenofibrate (micronized), fluvastatin sodium, gemfibrozil, lovastatin, nicotinic acid, Pravastatin sodium and Simvastatin at least a.At least a miscellany cardiovascular drug can be selected from ReoPro, prostaglandin E1, hydrochloric acid arbutamine, arbutamine, clopidogrel bisul-phate, dipyridamole, eptifibatide, Midodrine Hydrochloride, pentoxifylline, Ticlopidine Hydrochloride and Tirofiban hydrochloride at least a.(referring to 215-336 page or leaf) as Nursing 2001Drug Handbook.
At least a non-narcotic analgesics or febrifuge can be selected from Paracetamol, acetylsalicylic acid, choline magnesium trisalicylate, Diflonid and magnesium salicylate at least a.At least a nonsteroidal anti-inflammatory agent can be selected from celecoxib, Potassium diclofenac, Diclofenac sodium, R-ETODOLAC, fenoprofen calcium, U-27182, Ibuprofen BP/EP, indomethacin, indomethacin sodium trihydrate, Ketoprofen, ketorolac tromethamine, Maxicom, naprosine, naproxen sodium, Evil promazine, piroxicam, rofecoxib and sulindac at least a.At least a narcotic or opium anodyne can be to be selected from alfentanil hydrochloride, hydrochloric acid Bu Bennuo phenol, butorphanol tartrate, codeine phosphate, codeine sulfate, fentanyl citrate, Fentanyl Transdermal System, transform the fentanyl of slilme, the hydrochloric acid hydromorphone, isonipecaine hydrochloride, methadone hydrochloride, Srm-Rhotaard, morphine sulfate, morphine tartrate, nalbuphlne hydrochloride, oxycodone hydrochloride, the pectic acid oxycodone, oxymorphone hydrochloride, pentazocine hydrochloride, pentazocine hydrochloride and naloxone hydrochloride, pentazocine lactate, regretol, propoxyphene napsylate, remifentanil hydrochloride, sufentanil citrate and tramadol hydrochloride at least a.At least a sedative hypnotic can be selected from Chloral Hydrate, estazolam, flurazepam hydrochloride, Sodital, vetanarcol, sodium phenobarbital, seotal, temazepam, triazolam, Zaleplone and zolpidem tartrate at least a.At least a anticonvulsive agent can be selected from sodium acetazolamide, carbamazepine, clonazepam, chlorazepate, diazepam, divalproex sodium, ethosuximide, fosphenytoin sodium, gabapentin, lamotrigine, sal epsom, phenylethyl barbituric acid, sodium phenobarbital, Phenytoin Sodium Salt, phenytoin Sodium, phenytoin Sodium (long-term usefulness), Primidone, hydrochloric acid Tiagabine, topiramate, Sodium Valproate and valproic acid at least a.At least a thymoleptic can be to be selected from Warner), amitriptyline embonate, Omnipress, bupropion hydrochloride, citalopram hydrobromate, clomipramine hydrochloride, the hydrochloric acid desipramine, adapin, fluoxetine Hydrochloride, imipramine hydrochloride, imipramine embonate, Zispin, the hydrochloric acid nefazodone, hydrochloric acid Ka Jia is for woods, paroxetine hydrochloride, phenelzine sulfate, sertraline hydrochloride, tranylcypromine sulfate, trimipramine maleate and VENLAFAXINE HCL at least a.At least a anxiolytic can be selected from alprazolam, buspirone hydrochloride, bent, chlordiazepoxide, chlorazepate, diazepam, doxepin hydrochloride, Paxistil, hydroxyzine hydrochloride, Paxistil, lorazepam, meprobamate, midazolam hydrochloride and oxazepam at least a.At least a antipsychotic drug can be to be selected from Torazina, W-108, fluphenazin decanoate, fluphenazine enanthate, fluphenazine hydrochloride, haloperidol, haloperidol decanoate, the lactic acid haloperidol, loxapine hydrochloride, loxapine succinate, mesoridazine besilate, Molindone Hydrochloride, olanzapine, Trilifan, pimozide, prochlorperazine, quetiapine fumarate, Risperidone, mellaril, thiothixene, thiothixene hydrochloride and triphthasine at least a.At least a central nervous system stimulant can be selected from amfetamine sulfate, caffeine, dextro-amphetamine sulfate, doxapram hydrochloride, methamphetamine hydrochloride, Methylphenidylacetate, modafinil, pemoline and phentermine hydrochloride at least a.At least a antiparkinsonian drug can be selected from Amantadine Hydrochloride, benztropine mesylate, biperiden hydrochloride, biperiden lactate, bromocriptine methanesulfonate, carbidopa and levodopa, Entacapone, levodopa, pergolide mesilat, two hydrochloric acid pramipexoles, ropinirole hydrochloride, SelegilineHydrochloride, tolcapone and Benzhexol HCL at least a.At least a miscellany central nervous system medicine can be selected from bupropion hydrochloride, donepezil hydrochloride, Droperidol, fluvoxamine maleate, Quilonum Retard, Lithium Citrate, Naratriptan hydrochloride, polacrilex,nicotine, Nicotine transdermal system, Rapinovet, rizatriptan benzoate, Sibutramine Hydrochloride monohydrate, Sumatriptan Succinate, romotal and Zomitriptan at least a.(referring to 337-530 page or leaf) as Nursing 2001 Drug Handbook.
At least a cholinergic agent (parasympathomimetic agent) can be selected from bethanechol chloride, edrophonium chloride, Vagostigmine bromide, methyl-sulfuric acid prostigmin(e), eserine salicylate and pyridostigmine bromide at least a.At least a anticholinergic can be selected from Tropintran, bentrl hydrothloride, robinul, tropine, hyoscyamine sulfate, probanthine, scopolamine, Scopolamine Butylbromide and scopolamine hydrobromide at least a.At least a adrenergic agent (sympathomimetic) can be selected from Dobutamine Hydrochloride USP, dopamine hydrochloride, Metaraminol Bitartrate, tartrate nor-epinephrine, phenylephrine hydrochloride, pseudoephedrine hydrochloride and pseudoephedrine sulfate at least a.At least a adrenergic blocking drug (sympathicolytic) can be selected from Dihytamine, gynergen, desernil and propranolol hydrochloride at least a.At least a skeletal muscular relaxant can be selected from baclofen, Somalgit, benzoflex, cyclobenzaprine hydrochloride, Dantrolene Sodium, methocarbamol and tizanidine hydrochloride at least a.At least a neuromuscular blocking agents can be selected from atracurium, cisatracurium besylate, doxacurium, mivacurium chloride, pancuronium bromide, pipecuronium bromide, rapacuronium bromide, Zemuron, Choline Chloride Succinate, tubocurarine chloride and org NC45 at least a.(referring to 531-84 page or leaf) as Nursing 2001 Drug Handbook.
At least a antihistaminic can be selected from brompheniramine maleate, cetirizine hydrochloride, chlorpheniramine maleate, tavehil, Periactinol hydrochloride, Vena, fexofenadine hydrochloride, Loratadine, promethazine hydrochloride, promethazine teoclate and triprolidine hydrochloride at least a.At least a bronchodilator can be to be selected from salbutamol, albuterol sulfate, aminophylline, Tropintran, ephedrine sulfate, suprarenin, adrenaline acid tartrate, adrenalin hydrochloride, ipratropium bromide, Racemic isoproterenol, Vapo-Iso, isoproterenol sulfate, levalbuterol hydrochloride, metaproterenol sulfate, Zy 15061, pirbuterol acetate, salmeterol xinafoate, terbutaline sulfate and Zy 15061 at least a.At least a expectorant or cough medicine can be selected from benzonatate, codeine phosphate, codeine sulfate, dextromethorphan HBr, Vena, guaiacol glycerol ether and hydromorphone at least a.At least a miscellany respiratory system drug can be selected from acetylcysteine, beclomethasone dipropionate, Surfactant TA, budesonide, exogenous pulmoalveolar surfactant (calfactant), Sodium Cromoglicate, dornase alfa, U-53217A, flunisolide, fluticasone propionate, Menglusitena, sodium nedocromil, palivizumab, the third scorching pine, Zafirlukast and Zileuton at least a.(referring to 585-642 page or leaf) as Nursing 2001 DrugHandbook.
At least a antacid, sorbent material or antiflatulent can be selected from aluminium carbonate, aluminium hydroxide, lime carbonate, Ripon, magnesium hydroxide, magnesium oxide, Simethicone and sodium bicarbonate at least a.At least a digestive ferment or gallbladdergallstonecholetithiasis solubilizing agent can be selected from pancreatin, steapsin and ursodesoxycholic acid at least a.At least a diarrhea can be selected from attapulgite, bismuth subsalicylate, polycarbophil calcium, diphenoxylate hydrochloride and Tropintran, Loperamide, Sostatin LAR, tinctura opii and tinctura opii (containing camphor) at least a.At least a laxative can be selected from bisacodyl (bisocodyl), polycarbophil calcium, Sacred bark, Sacred bark aromatic fluid extract, Sacred bark fluid extract, Viscotrol C, dioctyl calcium sulfosuccinate, Docusate Sodium, glycerine, lactulose, magnesium citrate, magnesium hydroxide, sal epsom, methylcellulose gum, mineral oil, polyoxyethylene glycol or electrolyte solution, psyllium, sennae and sodium phosphate at least a.At least a antiemetic can be selected from Torazina, umine, dolasetron mesilate, dronabinol, Granisetron Hydrochloride, Ancolan, metoclopramide hydrochloride, ondansetron hydrochloride, Trilifan, SKF-4657, prochlorperazine edisylate, prochlorperazine maleate, promethazine hydrochloride, Scopolamine, thiethylperazine dimaleate and trimethobenzamide hydrochloride at least a.At least a anti-ulcerative drug can be selected from cimitidine, cimetidine hydrochloride, famotidine, Lansoprazole, colloidal bismuth subcitrate, nizatidine, omeprazole, Sodium rabeprazole, ranitidine bismuth citrate, ranitidine hydrochloride and sucralfate at least a.(referring to 643-95 page or leaf) as Nursing 2001 Drug Handbook.
At least a cortin can be to be selected from Betamethasone Valerate, betamethasone acetate or betamethasone sodium phosphate, betamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisone acetate, hydrocortisone, hydrocortisone acetate, the hydrocortisone cipionate, the hydrocortisone sodium phosphate, hydrocortisone sodium succinate, medrat, Methylprednisolone Acetate, Methylprednisolone Sodium Succinate, Ultracortene-H, predniso lone acetate, prednisolone phosphate sodium, prednisolone 21-tertbutylacetate, prednisone, triamcinolone, the third scorching pine and triamcinolone diacetate at least a.At least a male sex hormone or anabolic steroid can be selected from the different azoles of alkynes hydroxyl androstene, FL, methyltestosterone, abolon, Nrolone Phenylpropionate, testosterone, depo-testosterone, Testoviron-Depot, testosterone propionate and transdermal testosterone system at least a.At least a oestrogenic hormon or progestin can be to be selected from esterified estriol, estradiol, estradiol cypionate, estradiol/norethindrone acetate transdermal system, estradiol valerate, oestrogenic hormon (puting together), estropipate, ethinylestradiol, ethinylestradiol and desogestrel, ethinylestradiol and Norethindrone diacetate, ethinylestradiol and desogestrel, ethinylestradiol and Norethindrone diacetate, ethinylestradiol and Levonorgestrel, ethinylestradiol and Norethisterone, ethinylestradiol and Norethisterone Acetate, ethinylestradiol and vinylestrenolone, ethinylestradiol and methylnorethindron, ethinylestradiol and Norethisterone Acetate and ferrous fumarate, Levonorgestrel, medroxyprogesterone acetate, norquen mestranol and Norethisterone, Norethisterone, Norethisterone Acetate, methylnorethindron and progesterone at least a.At least a chorionic gonadotrophin can be selected from ganirelix acetate, acetic acid gonadorelin, histrelin acetate and Menotropins at least a.At least a antidiabetic drug or glucagon can be selected from Acarbose, P-607, glimepiride, Glipizide, hyperglycemic-glycogenolytic factor, Glyburide, Regular Insulin, Walaphage, miglitol, hydrochloric acid U-721017E, repaglinide, rosiglitazone maleate and troglitazone at least a.At least a Triiodothyronine can be selected from levothyroxine sodium, Cyronine, liotrix and thyroid powder at least a.At least a Triiodothyronine antagonist can be to be selected from Thiamazole, potassiumiodide, potassiumiodide (saturated solution), propylthiouracil, radioiodine (sodium iodide 131I) and Lugol's solution at least a.At least a pituitrin can be selected from thyroliberin, cosyntropin, desmopressin acetate, leuprorelin acetate, long-acting thyroliberin, somatrem, tethelin and Hou Yejiayasu at least a.At least a parathyroid gland sample medicine can be selected from U-32070E, thyrocalcitonin (people), thyrocalcitonin (salmon), calcitriol, dihydrotachysterol and etidronate disodium at least a.(referring to 696-796 page or leaf) as Nursing2001 Drug Handbook.
At least a diuretic(s) can be selected from acetazolamide, sodium acetazolamide, Amiloride, bumetanide, chlorthalidone, ethacrynate sodium, Uregit, Furosemide, hydrochlorothiazide, phenylene blue, mannitol, methaqualone, spironolactone, torsemide, triamterene and urea at least a.At least a electrolytic solution or substitutional solution can be to be selected from lime acetate, lime carbonate, calcium chloride, citrate of lime, Neo-Calglucon, Glucoheptonic Acid Calcium salt, calglucon, calcium lactate, calcium phosphate (bibasic), calcium phosphate (triple-substituted), dextran (high molecular), dextran (lower molecular weight), hydroxyethylamyle, magnesium chloride, sal epsom, potassium acetate, saleratus, Repone K, Potassium Gluconate, ringer's inj, ringer's inj (lactic acid) and sodium-chlor at least a.At least a souring agent or basifier can be selected from sodium bicarbonate, Sodium.alpha.-hydroxypropionate and tromethane at least a.(referring to 797-833 page or leaf) as Nursing 2001 Drug Handbook.
At least a hematopoietic can be selected from Ferrous Fumarate, Ferrous Gluconate, ferrous sulfate, ferrous sulfate (dried), Iron Dextran, iron sorbitol citrate, polysaccharide-iron complex and Sodium ferric gluconate mixture at least a.At least a anti-coagulant can be selected from Ardeparin Sodium, dalteparin sodium, Danaparoid sodium, Enoxaparin Sodium, calciparine, heparin sodium and Warfarin at least a.At least a blood derivatives can be selected from 5% albumin, 25% albumin, antihemophilic factor, antisuppressor coagulant mixture, antifibrinolysin EU (people), the IX factor (people), IX factor mixture and plasma protein fraction at least a.At least a thrombolytic enzymes can be selected from alteplase, anistreplase, reteplase (recombinant chou), streptokinase and urokinase at least a.(referring to 834-66 page or leaf) as Nursing2001 Drug Handbook.
At least a alkanisation medicine can be to be selected from busulfan, carbon platinum, carmustine, Chlorambucil, cis-platinum, endoxan, ifosfamide, Lomustine, mustine hydrochlcride, melphalan, hydrochloric acid melphalan, streptozocin, Temozolomide and plug to replace at least a of group.At least a metabolic antagonist can be selected from capecitabine, CldAdo, cytosine arabinoside, 5-fluodeoxyuridine, fludarabine phosphate, Fluracil, hydroxyurea, purinethol, methotrexate, methotrexate sodium and Tioguanine at least a.At least a microbiotic antitumor agent can be selected from bleomycin sulfate, gengshengmeisu, DaunoXome, daunorubicin hydrochloride, doxorubicin hydrochloride, hydrochloric doxorubicin liposome, epirubicin hydrochloride, idarubicin hydrochloride, mitomycin, pentostatin, Plicamycin and alrubicin at least a.The antitumor agent of at least a change hormonal equilibrium can be selected from arna holder (department) azoles, bicalutamide, estramustine phosphate sodium, Exemestane, Drogenil, Goserelin Acetate, letrozole, leuprorelin acetate, Magace, Nilutamide, Tamoxifen Citrate, testolactone and Toremifene Citrate at least a.At least a miscellany antitumor agent can be to be selected from asparaginase, bacille Calmette-Guerin vaccine (BCG) (intravesical is effective), dacarbazine, many Xi Taqi, etoposide, the phosphoric acid Zuyeyidal, gemcitabine hydrochloride, irinotecan hydrochloride, mitotane, mitoxantrone hydrochloride, taxol, Pegaspargase, porfimer sodium, procarbazine hydrochloride, Rituximab, teniposide, topotecan hydrochloride, trastuzumab, vitamin A acid, Vinblastine sulphate, vincristine sulphate and vinorelbine tartrate at least a.(referring to 867-963 page or leaf) as Nursing 2001 Drug Handbook.
At least a immunosuppressor can be to be selected from azathioprine, basiliximab, ciclosporin, to reach at least a of (gram) pearl monoclonal antibody, lymphocyte immune globulin, Orthoclone OKT 3, mycophenolate mofetil, hydrochloric acid mycophenolate mofetil, sirolimus and tacrolimus.At least a vaccine or toxoid can be diphtheria and tetanus toxid-acellular pertussis vaccine, diphtheria and tetanus toxid-whole cell pertussis vaccine, B-mode influenzae conjugate vaccine, Hepatitis A Vaccine (deactivation), hepatitis B vaccine (recombinant chou), the influenza virus vaccine 1999-2000 trivalent type A ﹠amp that is selected from BCG vaccine, Cholera Vaccine, diphtheria and tetanus toxid (absorption), absorption; B (surface antigen of purifying), influenza virus vaccine 1999-2000 trivalent type A ﹠amp; B (subvirral particle of subvirral particle or purifying), influenza virus vaccine 1999-2000 trivalent type A ﹠amp; B (totivirus particle), japanese encephalitis virus vaccine (deactivation), ImuLyme (recombinant chou OspA), measles-mumps-rubella virus vaccine (living), measles-mumps-rubella virus vaccine (attenuation of living), measles virus vaccines (attenuation of living), meningococcal polysaccharide vaccine, mumps virus vaccine (living), plague vaccine, Pnu-Imune 23 (multivalence), poliovirus vaccine (deactivation), poliovirus vaccine (is lived, oral, trivalent), Rabies Vaccine (absorption), Rabies Vaccine (human diploid cell), rubella-mumps virus vaccine (living), rubella virus vaccine (is lived, attenuation), Toxoid,tetanus (absorption), Toxoid,tetanus (liquid), antityphoid vaccine (oral), antityphoid vaccine (parenteral injection is used), Typhoid Vi Polysaccharide Vaccine, Varivax and Yellow Fever Vaccine at least a.At least a toxinicide or antivenin can be selected from black widow spider antivenin, rattlesnake antivenin (multivalence), Diphtheria Antitoxin (horse) and little tail Naja antivenin at least a.At least a immune serum can be to be selected from cytomegalovirus immune globulin (intravenous), hepatitis B virus immunoglobulin (Ig) (people), the immunoglobulin (Ig) of intramuscularly, intravenous immunoglobulin (Ig), rabies immune globulin (people), intravenous respiratory syncytial virus immune globulin (people), Rh 0(D) immunoglobulin (Ig) (people), intravenous Rh 0(D) immunoglobulin (Ig) (people), tetanus immune globulin (people) and varicella zoster immune globulin is at least a.At least a biological answer-reply instrumentality can be selected from rIL-2, recombinant human erythropoietin, filgrastim, the acetic acid glatiramer that is used to inject, interferon alfacon-1, Intederon Alpha-2a (recombinant chou), Interferon Alpha-2b (recombinant chou), interferon beta-1a, interferon beta-1b (recombinant chou), gamma interferon 1-b, R-12564, oprelvekin and sargramostim at least a.(referring to 964-1040 page or leaf) as Nursing 2001 DrugHandbook.
At least a eye can be selected from bacitracin, paraxin, ciprofloxacin HCl, Abboticine, Gentamicin Sulfate, 0.3% Ofloxacine USP 23, Polymyxin B-sulfate USP, 10% Sulf-10,15% Sulf-10,30% Sulf-10, tobramycin and vidarabine at least a with anti-infective.At least a eye can be selected from dexamethasone, dexamethasone sodium phosphate, 0.1% Diclofenac Sodium, flurandrenolide, Sodium flurbiprofen, ketorolac tromethamine, prednisolone (suspension) and prednisolone phosphate sodium (solution) at least a with antiphlogiston.At least a myotic can be selected from Ovisot, carbachol (eye drops), carbachol (topical), diethoxyphosphinylthiocholine, pilocarpine, hydrochloric acid pilocarpine and Pilopt-N at least a.At least a iridodilator can be selected from Tropintran, cyclopentolate HCL 99, adrenalin hydrochloride, epinephryl borate, homatropine hydrobromide, phenylephrine hydrochloride, scopolamine hydrobromide and mydriacyl at least a.At least a eye can be selected from naphcon, oxymetazoline hydrochloride and tetrahydrozoline hydrochloride at least a with vasoconstrictor.At least a miscellany medicament for the eyes can be selected from least a of aplonidine hydrochloride, betaxolol hydrochloride, brimonidine tartrate, carteolol hydrochloride, dipivefrin hydrochloride, dorzolamide hydrochloride, emedastine difumarate, uranine, ketotifen, latanoprost, Levobunolol Hydrochorid, hydrochloric acid metipranolol, sodium-chlor (height oozes) and timolol maleate.At least a ear medicine can be selected from boric acid, Urea Peroxide, paraxin and trolamine polypeptide oleic acid condensation product at least a.At least a nose medicine can be at least a (referring to the 1041-97 page or leaf as Nursing 2001 DrugHandbook) that is selected from beclomethasone dipropionate, budesonide, ephedrine sulfate, adrenalin hydrochloride, flunisolide, fluticasone propionate, naphcon, oxymetazoline hydrochloride, phenylephrine hydrochloride, tetrahydrozoline hydrochloride, the third scorching pine and xylometazoline hydrochloride.
At least a part can be to be selected from aciclovir with anti-infective, amphotericin B, the nonane diacid emulsifiable paste, bacitracin, Nitric acid butoconazole, cleocin phosphate, clotrimazole, econazole, Abboticine, Gentamicin Sulfate, KETOKONAZOL, mafenide acetate, metronidazole (local using), miconazole nitrate, mupirocin, naftifine hydrochloride, neomycinsulphate, nitrofural, nysfungin, Sulfadiazine Silver, Terbinafine Hcl, Triaconazole, tetracycline hydrochloride, it is at least a that tioconazole and tinea capitis are moved back.At least a Scabicide or pediculicide can be selected from crotamiton, lindane, permethrin and pyrethrin at least a.At least a topical corticosteroid can be selected from Sch-11460, Valisone, Clobesol, desonide, desoximetasone, dexamethasone, dexamethasone sodium phosphate, diflorasone diacetate, Metosyn, fluocinolone acetonide, Cordran, fluticasone propionate, halcionide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyric ester, valeric acid hydrocortisone, Sch-32088 and Triamcinolone Acetonide at least a.(referring to 1098-1136 page or leaf) as Nursing 2001 DrugHandbook.
At least a VITAMIN or mineral substance can be to be selected from vitamin A, vitamin B complexes, cyanocobalamin, folic acid, hydroxocobalamine, calcium leucovorin, nicotinic acid, niacinamide, pyridoxine hydrochloride, riboflavin, VITMAIN B1, vitamins C, vitamins D, cholecalciferol, ergocalciferol, novel vitamin D analogues, the degree ostelin, Zemplar, vitamin-E, the vitamin K analogue, vitamin K1, Sodium Fluoride, sodium-chlor (local using), trace elements, chromium, copper, iodine, manganese, selenium and zinc at least a.At least a heat material can be selected from amino acid infusion agent (crystalline), be in amino acid infusion agent in the glucose, have the agent of electrolytical amino acid infusion, be in the glucose have the agent of electrolytical amino acid infusion, be used for liver failure the agent of amino acid infusion, be used for hypermetabolism stress the agent of amino acid infusion, be used for the agent of amino acid infusion, glucose, fat emulsion and medium chain triglyceride at least a of renal failure.(referring to 1137-63 page or leaf) as Nursing 2001 Drug Handbook.
Anti-IL-6 antibodies composition of the present invention can further comprise at least a being fit to arbitrarily and the composition or the pharmaceutical composition of significant quantity, described composition or pharmaceutical composition comprise and the such adjusting of needs, the cell of handling or treating, tissue, organ, animal or patient contact or are applied to its at least a anti-IL-6 antibodies, randomly further comprise be selected from least a TNF antagonist (as but be not limited to TNF chemistry or protein antagonist, TNF mono-clonal or polyclonal antibody or fragment, soluble TNF acceptor is (as p55, p70 or p85) or fragment, their fusion polypeptide, or small molecules TNF antagonist, as conjugated protein I of TNF or II (TBP-I or TBP-II), nerelimomab, infliximab, etanercept, CDP-571, CDP-870, Afelimomab, Lenercept etc.), antirheumatic is (as methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, disodium aurothiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), muscle relaxant, narcotic, NSAID (non-steroidal anti-inflammatory drug) (NSAID), anodyne, narcotic, tranquilizer, local anesthetic, neuromuscular blocking agents, biocide is (as glucosaminide, antifungal drug, antiparasitic, antiviral drug, carbon (mixing) penem, cynnematin, fluoroquinolone, macrolide, penicillin, sulfanilamide (SN), tsiklomitsin, other biocides), antipsoriatic, corticosteroid, anabolic steroid, the medicine that diabetes are relevant, mineral substance, nutrition, thyroid, VITAMIN, the hormone that calcium is relevant, diarrhea, cough medicine, antiemetic, anti-ulcerative drug, laxative, anti-coagulant, erythropoietin (as the epoetin), filgrastim is (as G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunization, immunoglobulin (Ig), immunosuppressor is (as basiliximab, ciclosporin, reach (gram) pearl monoclonal antibody), tethelin, the hormone replacement medicine, estrogenic agents, iridodilator, cycloplegic, alkylating agent, antimetabolite, mitotic inhibitor, radioprotector, thymoleptic, antimaniacal drugs, antipsychotic drug, antianxiety agent, soporific, sympathomimetic, stimulant, E2020, tacrine, asthma drug, beta-agonists, the steroid class that sucks, the leukotrienes inhibitor, methyl xanthine, Cromoglycic Acid, suprarenin or analogue, α streptodornase (Pulmozyme), cytokine or cytokine antagonist at least a.The nonrestrictive example of such cytokine includes but not limited to any (as IL-1, the IL-2 etc.) of IL-1 to IL-23.The dosage that is fit to is well-known in the art.Referring to as Wells etc., compile Pharmacotherapy Handbook, the 2nd edition, Appletonand Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon PocketPharmacopoeia 2000, senior version, Tarascon Publishing, Loma Linda, CA (2000), each comfortable this is incorporated herein by reference fully.
Anticancer or anti-infective so also can comprise with at least a antibody of the present invention associate, combine, the lps molecule of preparation or administration simultaneously jointly.Toxin can randomly work and kill pathologic cell or tissue with selectivity.Pathologic cell can be cancer cells or other cells.Such toxin can be but be not limited to toxin purifying or reorganization or comprise the toxin fragment of the functional cell toxicity structural domain of at least one toxin, as be selected from least a Ricin, diphtheria toxin, snake venom or bacteriotoxin.The term toxin also comprises any naturally occurring, sudden change or the bacterium of reorganization or intracellular toxin and the extracellular toxin that virus is produced, and described bacterium or virus can cause people or other Mammals pathology situations, comprise causing dead toxin shock.Such toxin can include but not limited to enterotoxigenic coli heat enterotoxin (LT), heat-stable toxin (ST), will he cytotoxin, Aeromonas enterotoxin, toxic shock syndrome toxin-1 (TSST-I), staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB) or staphyloentero-toxin C (SEC), suis enterotoxin etc.Such bacterium includes but not limited to enterotoxigenic intestinal bacteria (ETEC), the intestinal bacteria of enterorrhagia (as the 0157:H7 serological type strain), staphylococcus is (as streptococcus aureus (Staphylococcus aureus), staphylococcus pyogenes (Staphylococcus pyogenes)), Shigellae is (as shigella dysenteriae (Shigelladysenteriae), shigella flexneri (Shigella flexneri), Shigellae (Shigella sonnei) in Bo Yide Shigellae (Shigella boydii) and the Song), Salmonellas is (as salmonella typhi (Salmonella typhi), Salmonella choleraesuls (Salmonella cholera-suis), Salmonella enteritidis (Salmonella enteritidis)), clostridium is (as bacillus aerogenes capsulatus (Clostridium perfringens), difficulty is distinguished clostridium (Clostridiumdificile), clostridium botulinum (Clostridium botulinum)), Campylobacter is (as campylobacter jejuni (Camphlobacter jejuni), embryo's Campylobacter (Camphlobacterfetus)), Helicobacter pylori (as helicobacter pylori (Heliobacter pylori)), Aeromonas is (as Aeromonas sobria (Aeromonas sobria), Aeromonas hydrophila (Aeromonas hydrophila), Aeromonas caviae (Aeromonas caviae)), Plesiomonas shigelloides (Pleisomonasshigelloides), small intestine colon yersinia enterocolitica (Yersina enterocolitica), vibrios is (as vibrio cholerae (Vibrios cholerae), Vibrio parahemolyticus (Vibrios parahemolyticus)), Cray Bai Shi bacillus, Pseudomonas aeruginosa (Pseudomonas aeruginosa) and streptococcic bacterial strain.Referring to as Stein, compile INTERNAL MEDICINE, the 3rd edition, pp 1-13, Little, Brownand Co., Boston, (1990); Evans etc. compile Bacterial Infections of Humans:Epidemiology and Control, the 2nd edition, pp 239-254, Plenum Medical BookCo., New York (1991); Mandell etc., Principles and Practice of InfectiousDiseases, the 3rd edition, Churchill Livingstone, New York (1990); Berkow etc. compile The Merck Manual, the 16th edition, Merck and Co., Rahway, NJ., 1992; Wood etc., FEMSMicrobiology Immunology, 76:121-134 (1991); Marrack etc., Science, 248:705-711 (1990), content is incorporated herein by reference fully at this separately.
Anti-IL-6 antibodies compound of the present invention, composition or combination can further comprise at least a any proper assistant, such as but not limited to thinner, tackiness agent, stablizer, buffer reagent, salt, lipophilic solvent, sanitas, adjuvant etc.Pharmaceutically acceptable auxiliary agent is preferred.The nonrestrictive example for preparing the method for such sterile solution is well-known in the art, such as but not limited to Gennaro, compiles, Remington ' s Pharmaceutical Sciences, the 18th edition, Mack Publishing Co. (Easton, PA) 1990.As known in the art or described herein, can select to be suitable for administering mode routinely, the pharmaceutically acceptable carrier of the solvability of anti-IL-6 antibodies, fragment or variant compositions and/or stability.
Be used for that the pharmaceutical excipient of the present composition and additive comprise but the albumen, peptide, amino acid, lipid and the carbohydrate that are not limited to exist alone or in combination (as carbohydrate, comprise monose, disaccharides, trisaccharide, tetrose and oligosaccharides; Deutero-carbohydrate, for example carbohydrate of sugar alcohol, glyconic acid, esterification etc.; And polysaccharide or glycopolymers), form separately by weight or volumeter be contained in the combination with 1-99.99%.Representational albumen vehicle comprises serum albumin, for example human serum albumin (HSA), recombination human serum albumin (rHA), gelatin, casein etc.Representational amino acid/the antibody component that can also play the buffer capacity effect comprises L-Ala, glycine, arginine, trimethyl-glycine, Histidine, L-glutamic acid, aspartic acid, halfcystine, Methionin, leucine, Isoleucine, Xie Ansuan, methionine(Met), phenylalanine, aspartame sugar etc.A kind of preferred amino acids is a glycine.
Be applicable to that carbohydrate vehicle of the present invention comprises, for example, such as the monose of fructose, maltose, semi-lactosi, glucose, D-seminose, sorbose etc.; Disaccharides such as lactose, sucrose, trehalose, cellobiose etc.; Polysaccharide such as raffinose, melizitose, Star Dri 5, dextran, starch etc.; And such as the sugar alcohol of mannitol, Xylitol, maltose alcohol, Saccharum lactis, Xylitol Sorbitol Powder (sorbyl alcohol), inositol etc.Carbohydrate vehicle preferred for the present invention is mannitol, trehalose and raffinose.
The anti-IL-6 antibodies composition also can comprise buffer reagent or pH regulator agent; Typically, buffer reagent is the salt of preparation from organic acid or alkali.Representational buffer reagent comprises organic acid salt, for example citrate, ascorbate salt, gluconate, carbonate, tartrate, succinate, acetate or phthalate; Tris, tromethane hydrochloride or phosphate buffer.The preferred reducing agents that is used for the present composition is an organic acid salt, for example Citric Acid.
In addition, anti-IL-6 antibodies composition of the present invention can comprise polymeric excipient/additive, for example polyvinylpyrrolidone, ficoll (glycan), dextrates are (as cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin for example), polyoxyethylene glycol, seasonings, biocide, sweetener, antioxidant, static inhibitor, tensio-active agent (as polysorbate, for example " polysorbas20 " and " tween 80 "), lipid (as phosphatide, lipid acid), steroid (as cholesterol) and sequestrant (as EDTA).
These and other are known to be applicable to that pharmaceutical excipient and/or additive according to anti-IL-6 antibodies of the present invention, part or variant compositions are known in the art, as lists in ＂ Remington:The Science ﹠amp; Practice of Pharmacy ＂, the 19th edition, Williams ﹠amp; Williams, (1995) and ＂ Physician ' s Desk Reference ＂, the 52nd edition, MedicalEconomics, Montvale, among the NJ (1998), its content is incorporated herein by reference fully at this.Preferred carrier or excipient material are carbohydrate (as carbohydrate and sugar alcohol) and buffer reagent (as Citrate trianion) or polymeric reagent.The carrier molecule of making illustration is to can be used for mucopolysaccharide, the hyaluronic acid that intraarticular is sent.
Prescription
As mentioned above, the invention provides the stable prescription that comprises at least a anti-IL-6 antibodies that is in the pharmaceutically acceptable prescription, it preferably comprises the phosphate buffer with salt solution or selected salt and preserves solution, with the prescription that contains sanitas, and be suitable for multi-usage preservation prescription medicinal or that the animal doctor uses.Preserving fills a prescription to contain is at least a known sanitas in the aqueous diluent or randomly is selected from least a phenol, meta-cresol, p-cresol, ortho-cresol, parachlorometacresol, phenylcarbinol, phenylmercuric nitrous acid ester, Phenoxyethanol, formaldehyde, butylene-chlorohydrin, magnesium chloride (as hexahydrate), alkyl metagin (methyl, ethyl, propyl group, butyl etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and Thiomersalate, polymkeric substance or their mixture.As known in the art, can use any suitable concentration or mixing, for example about 0.0015%, or wherein any scope, value or mark.Nonrestrictive example comprises, does not contain sanitas, about 0.1-2% meta-cresol is (as 0.2,0.3.0.4,0.5,0.9,1.0%), about 0.1-3% phenylcarbinol is (as 0.5,0.9,1.1,1.5,1.9,2.0,2.5%), about 0.001-0.5% Thiomersalate is (as 0.005,0.01), about 0.001-2.0% phenol is (as 0.05,0.25,0.28,0.5,0.9,1.0%) 0.0005-1.0% alkyl metagin is (as 0.00075,0.0009,0.001,0.002,0.005,0.0075,0.009,0.01,0.02,0.05,0.075,0.09,0.1,0.2,0.3,0.5,0.75,0.9,1.0%) etc.
As mentioned above, the invention provides a kind of product that wrapping material and at least one contain at least a anti-IL-6 antibodies and the phial of the solution that randomly is in given buffer reagent in the aqueous diluent and/or sanitas that comprises, wherein said packing comprised indicate the label that such solution can be preserved in 1,2,3,4,5,6,9,12,18,20,24,30,36,40,48,54,60,66,72 hour or longer for some time.The present invention further comprises a kind of first phial that comprises wrapping material, contains freeze dried at least a anti-IL-6 antibodies, with second phial of the aqueous diluent that contains given buffer reagent or sanitas, wherein said wrapping material comprise and instruct the patient to rebuild at least a anti-IL-6 antibodies in aqueous diluent to preserve 24 hours or the label of the solution of longer for some time to form.
As described herein or known in the art, can be by comprising recombinant means from mammalian cell or transgenosis prepared product, but or purifying originate from other biological, produce according at least a anti-IL-6 antibodies used in the present invention.
The scope of at least a anti-IL-6 antibodies comprises the amount that reconstruction produces in the product of the present invention, if in wet/dry systems, then concentration is the about 1000mg/ml of about 1.0 μ g/ml-, although lower or higher concentration is exercisable, and depend on and the delivery vector of expection should be different from transdermal patch, lung, transmucosal or infiltration or micro pump method as solution formula.
Preferably, aqueous diluent randomly further comprises pharmaceutically acceptable sanitas.Preferred sanitas comprises those sanitass that are selected from phenol, meta-cresol, p-cresol, ortho-cresol, parachlorometacresol, phenylcarbinol, alkyl metagin (methyl, ethyl, propyl group, butyl etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and Thiomersalate or their mixture.The concentration of used sanitas is the concentration that is enough to produce antimicrobial effect in the prescription.Such concentration depends on selected sanitas and is easy to be determined by those skilled in the art.
Other vehicle as isotonic agent, buffer reagent, antioxidant and preservative enhancers, can be chosen wantonly and preferably be added in the thinner.Isotonic agent such as glycerine uses under concentration known usually.The preferred buffer reagent of physiology tolerance that adds is to provide the pH control of improvement.Prescription can cover pHs widely, and for example from the about pH 10 of about pH 4-, preferred range is the about pH 9 of about pH 5-, and most preferred scope is about 6.0-about 8.0.Preferably, prescription of the present invention has the pH of about 6.8-about 7.8.Preferred reducing agents comprises phosphate buffer, most preferably sodium phosphate, particularly phosphate buffered saline (PBS) (PBS).
Other additives, for example the pharmaceutically acceptable solubilizing agent resembles polysorbas20 (polyoxyethylene (20) Arlacel-20), polysorbate40 (polyoxyethylene (20) sorbitan monopalmitate), tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymer) and PEG (polyoxyethylene glycol) or nonionic surface active agent, for example polysorbate20 or 80 or poloxamer 184 or 188, Pluronic Polymkeric substance, other segmented copolymer, and sequestrant, for example EDTA and EGTA can randomly add in prescription or the composition and assemble to reduce.If pump or plastic containers are used to use prescription, then these additives are particularly useful.The tendency of protein aggregation has been alleviated in the existence of pharmaceutically acceptable tensio-active agent.
Can at least a anti-IL-6 antibodies and the sanitas blended method that is selected from phenol, meta-cresol, p-cresol, ortho-cresol, parachlorometacresol, phenylcarbinol, alkyl metagin (methyl, ethyl, propyl group, butyl etc.), benzalkonium chloride, Solamin, sodium dehydroacetate and Thiomersalate or their mixture that is in the aqueous diluent be prepared prescription of the present invention by comprising.Mixing of sanitas during dissolving that utilization is conventional and blending means are carried out at least a anti-IL-6 antibodies and be in aqueous diluent.For example, at least a anti-IL-6 antibodies that will be in the measured quantity in the buffered soln with the amount that is enough to provide albumen and sanitas expectation concentration combines to prepare suitable prescription with the sanitas of expectation in being in buffered soln.Those skilled in the art will recognize that the change of this method.For example, component interpolation order, temperature and pH when whether using extra additive, preparation prescription are all factors that can be optimized concentration and employed means of administration.
Prescription required for protection can be offered the patient as settled solution or as the binary phial; described binary phial comprises with containing water, being in sanitas and/or vehicle in the aqueous thinner, preferably a phial of the freeze dried at least a anti-IL-6 antibodies of second phial of phosphate buffered saline buffer and/or salt solution and selected salt reconstruction.The binary phial that single solution phial maybe needs to rebuild can repeatedly re-use, and can satisfy single or multiple round-robin patient treatments, and therefore can provide more current getable medicine for treatment method more easily.
The present invention's product required for protection has from the purposes of administration in 24 hours or longer for some time immediately.Therefore, the present invention's product required for protection offers the significant interests of patient.Therefore prescription of the present invention randomly safe storage allows that packaging label indicates solution and can preserve and/or use under about 2 ℃-Yue 40 ℃ temperature and keep proteic biological activity for a long time in 6,12,18,24,36,48,72 or 96 hours or longer time.If use anticorrosion thinner, then such label can comprise reach 1-12 month, usage period of half a year, a year and a half and/or 2 years.
Can mix the solution that the method that is at least a antibody in the aqueous diluent prepares at least a anti-IL-6 antibodies of the present invention by comprising.Use conventional dissolving and blending means to mix.For example, be enough to provide albumen and randomly the amount of sanitas or buffer reagent expectation concentration will be at least a antibodies of the measured quantity in water or the buffer reagent to prepare suitable thinner.Those skilled in the art will recognize that the change of this method.For example, component interpolation order, temperature and pH when whether using extra additive, preparation prescription are all factors that can be optimized concentration and employed means of administration.
Product required for protection can be offered the patient as settled solution or as the binary phial, described binary phial comprises a phial of the freeze dried at least a anti-IL-6 antibodies of rebuilding with second phial that contains aqueous thinner.The binary phial that single solution phial maybe needs to rebuild can repeatedly re-use, and can satisfy single or multiple round-robin patient treatments, and therefore can provide more current getable medicine for treatment method more easily.
Product required for protection can offer the patient indirectly by the binary phial of a phial that settled solution is provided or comprises the freeze dried at least a anti-IL-6 antibodies of rebuilding with second phial that contains aqueous thinner to pharmacy, clinic or other such mechanism and facility.If at least a antibody-solutions of a less part can one or many takes out human consumer and/or the patient who is used for transferring to less phial and offers them by pharmacy or clinic from big container, then in this case, the settled solution volume can reach one liter or even bigger.
The device that comprises single phial system of generally acknowledging comprises and is used to send solution pen-injected device, BD Pens for example, BD
Figure A200680024037D00611
Figure A200680024037D00612
Figure A200680024037D00613
Figure A200680024037D00614
Figure A200680024037D00615
With
Figure A200680024037D00616
Figure A200680024037D00617
Genotronorm
Figure A200680024037D00618
Humatro
Figure A200680024037D00619
Figure A200680024037D006110
Roferon
Figure A200680024037D006111
J-tip Needle-
Figure A200680024037D006114
Figure A200680024037D006115
Figure A200680024037D006116
As BectonDickensen (Franklin Lakes, NJ, www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com; Bioject, Portland, Oregon (www.bioject.com); National Medical Products, Weston Medical (Peterborough, UK, www.weston-medical.com), Medi-Ject Corp (Minneapolis, MN, Www.mediject.com), manufacturing or exploitation, and similarly be fit to device.The device that comprises binary phial system of generally acknowledging comprises those pen-injected systems that are used for for the cartridge case reconstruction freeze-dried drug of sending reconstruction solution, for example
Figure A200680024037D006117
The example of other suitable devices comprises prefilled syringe, automatic injector, needleless injector and needle-less intravenous fluids utensil.
The present invention's product required for protection comprises wrapping material.Wrapping material also provide the condition of using product except that the desired information of administration.For wet/dryed product of binary phial, wrapping material of the present invention offer the patient and rebuild at least a anti-IL-6 antibodies to form solution and the guidance of this solution of use in 2-24 hour or longer for some time in aqueous diluent.For the solution product of single phial, labeled marker such solution can in 2-24 hour or longer for some time, use.The present invention's product required for protection has the purposes of people's medicinal product.
Can be by comprising with at least a anti-IL-6 antibodies and selected buffer reagent, preferably, the phosphate buffer blending means that contains salt solution or selected salt prepares prescription of the present invention.Use conventional dissolving and blending means to carry out at least a anti-IL-6 antibodies and the mixing that is in the buffer reagent in the aqueous diluent.For example, at least a antibody that will be in the measured quantity in water or the buffer reagent with the amount that is enough to provide albumen and buffer reagent expectation concentration combines to prepare suitable prescription with the buffer reagent of expectation in being in water.Those skilled in the art will recognize that the change of this method.For example, component interpolation order, temperature and pH when whether using extra additive, preparation prescription are all factors that can be optimized concentration and employed means of administration.
Required for protection stable or preserve prescription and can be used as settled solution or offer the patient as the binary phial, described binary phial comprises a phial of the freeze dried at least a anti-IL-6 antibodies of rebuilding with second phial that contains the sanitas that is in the aqueous diluent or buffer reagent and vehicle.The binary phial that single solution phial maybe needs to rebuild can repeatedly re-use, and can satisfy single or multiple round-robin patient treatments, and therefore can provide more current getable medicine for treatment method more easily.
Except that the lyophilized powder settled solution that comprises antibody, can produce the prescription or the method for other stable anti-IL-6 antibodies.One of non-settled solution be the prescription that comprises the particle suspension, described particle is the composition that contains anti-IL-6 antibodies with variable-sized structure, known multiple particle is microballoon, particulate (microparticle), nanoparticle, millimicro ball or liposome.According to U.S.4, instruction in 589,330 can contact with polymkeric substance and nonaqueous phase by the water that will contain promoting agent, evaporate nonaqueous phase afterwards so that from the particles coalesce of water, thus generate such relative homogeneous that contains promoting agent, spheric granular recipe basically.According to U.S.4, the instruction in 818,542, can use contain promoting agent first mutually and be scattered in polymkeric substance in the continuous solvent, and from suspension, remove described solvent, thereby prepare small porous particle by lyophilize or dilution-extraction-precipitation.The polymkeric substance that preferably is used for such preparation is for being selected from gelatin, agar, starch, arabogalactan, albumin, collagen, polyglycolic acid, poly(lactic acid), glycollide-L (-) rac-Lactide, poly-(6-caprolactone), poly-(6-caprolactone-CO-lactic acid), poly-(6-caprolactone-CO-oxyacetic acid), poly-(beta-hydroxy-butanoic acid), poly-oxyethylene, polyethylene, poly-(alkyl-2-cyanoacrylate), poly-(hydroxyethyl methylacrylate), polymeric amide, poly-(amino acid), poly-(2-hydroxyethyl DL-l-asparagine), poly-(urea ester), natural or the synthetic multipolymer or the polymkeric substance of poly-(L-phenylalanine/ethylene glycol/1,6-diisocyanato-Hexane) and poly-(methyl methacrylate).Particularly preferred polymkeric substance is a polyester, for example polyglycolic acid, poly(lactic acid), glycollide-L (-) rac-Lactide, poly-(6-caprolactone), poly-(6-caprolactone-CO-lactic acid) and poly-(6-caprolactone-CO-oxyacetic acid).The solvent that is used for dissolve polymer and/or activeconstituents comprises: water, hexafluoroisopropanol, methylene dichloride, tetrahydrofuran (THF), hexane, benzene or Perfluoroacetone sesquialter hydrate.With second method of disperseing to contain the phase of activeconstituents mutually can comprise exert pressure described first by spray orifice to form droplet.
Can use to be different from freeze dried method generation dry powder formulation, for example by spraying drying or evaporating solvent extraction or precipitated crystal composition, afterwards a step or multistep are to remove moisture or non-aqueous solvent.At U.S.6, instructed the preparation of spray-dired antibody preparation in 019,968.Providing under respirable dry powder condition, can be in the antibody in the solvent and randomly the solution of vehicle or slurry produce dry powder composite based on antibody by spraying drying.Solvent can comprise polar compound, for example easily exsiccant water and ethanol.Can strengthen antibody stability by carrying out the operation of anaerobic spraying drying, for example under nitrogen blanket or by utilizing nitrogen as dry gas.The prescription of another kind of relatively dry is a large amount of porous microstructures to be scattered in dispersion thing in the suspension medium that comprises hydrogen fluoroalkane propellent usually according to the instruction among the WO 9916419.Utilize dispersion thing that metered dose inhaler can be stable with this to be applied to patient's lung.Buchi Ltd. or Niro Corp have made employed equipment in the spraying drying medicine commercial production.
Can be applied to the patient according to the present invention with being at least a anti-IL-6 antibodies stable or that preserve in prescription or the solution described herein by multiple delivering method, described delivering method comprises SC or IM injection as known in the art; Through skin, lung, transmucosal, implantation, osmotic pump, cartridge case, micro pump or be other means understood by one of ordinary skill in the art.
Treatment is used
The present invention also provides a kind of method as known in the art or at least a IL-6 diseases associated of cell described herein, tissue, organ, animal or patient that is used for regulating or treating, described method has been used at least a IL-6 antibody of the present invention, uses or exposing cell, tissue, organ, animal or patient as the IL-6 antibody that will treat significant quantity.The present invention also provides a kind of method that is used for regulating or treating at least a IL-6 diseases associated of cell, tissue, organ, animal or patient, described IL-6 diseases associated includes but not limited to, at least a obesity, immune correlated disease, cardiovascular disorder, transmissible disease, malignant diseases or sacred disease.
The present invention also provides a kind of and has been used for regulating or the treatment cell, tissue, organ, the method of the relevant immune correlated disease of at least a IL-6 among animal or the patient, the relevant immune correlated disease of described IL-6 includes but not limited at least a rheumatoid arthritis, juvenile rheumatiod arthritis (JRA), the juvenile rheumatoid arthritis of generalized seizure, arthritic psoriasis, ankylosing spondylitis, stomach ulcer, the negative joint disease of serum (reaction), osteoarthritis, osteolysis, the aseptic of surgery implants relaxes, inflammatory bowel, ulcerative colitis, systemic lupus erythematosus, lupus erythematosus,cutaneous, lupus nephritis, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wei Geneishi granuloma disease, sarcoidosis, testitis/vasotomy reversal procedures, allergy/atopic disorder, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis, graft, the organ-graft refection, graft versus host disease (GVH disease), systemic inflammatory response syndrome, sepsis syndrome, gram positive sepsis, gram negative sepsis, the negative Sepsis of culture, the fungi Sepsis, the neutrocytopenia heating, urosepsis, meningococcemia, wound/hemorrhage, burn, ionizing rays exposes, acute pancreatitis, adult respiratory distress syndrome, rheumatoid arthritis, alcohol inductive hepatitis, chronic inflammation pathology, sarcoidosis, CrohnShi pathology, sicklemia, diabetes, ephrosis, atopic disorder, allergy, rhinallergosis, ragweed fever, perennial rhinitis, conjunctivitis, endometriosis, asthma, rubella, systemic anaphylaxis, dermatitis, pernicious anemia, hemolytic disease, thrombopenia (disease), the transplant rejection of any organ or tissue, renal transplant rejection, heart transplantation is repelled, liver transplantation is repelled, transplantation of pancreas is repelled, lung transplant rejection, bone marrow transplantation (BMT) is repelled, SAR, cartilage transplantation repels, bone graft repels, small intestine transplantation repels, fetal thymus is implanted and is repelled, parathyroid transplantation repels, the heterograft of any organ or tissue repels, allograft rejection, anti-acceptor allergy, Graves disease, Reynolds (family name) disease, the Type B insulin-resistant diabetes, asthma, myasthenia gravis, antibody-mediated cytotoxicity, the allergy of DI type, POEMS syndrome (polyneuropathy, the organomegaly, incretopathy, MG and skin change syndrome), polyneuropathy, the organomegaly, incretopathy, MG, skin changes syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed connective tissue disease, primary A Disen (family name) disease, diabetes, chronic active hepatitis, primary biliary cirrhosis, leukodermia, vasculitis, cardiotomy syndrome behind the MI, IV type allergy, contact dermatitis, hypersensitivity pneumonitis, allograft rejection, because the granuloma of organism in the cell, drug allergy, metabolic disease/idiopathy, Wilson's disease, hemochromatosis, alpha-1-Antitrypsin deficiency, diabetic retinopathy, Hashimoto thyroiditis, osteoporosis, the hypothalmus-pituitary-adrenal axis assessment, primary biliary cirrhosis, thyroiditis, encephalomyelitis, emaciation, cystic fibrosis, newborn infant's chronic lung disease, chronic obstructive pulmonary disease (COPD), familial is bitten hemocyte lymphohistocysis disease (familial hematophagocyticlymphohistiocytosis), the dermatologic illness, psoriasis, bald, nephrotic syndrome, ephritis, glomerulonephritis, acute renal failure, hemodialysis, uremia, poison, preeclampsia, the okt3 therapy, anti-cd3 therapy, the cytokine therapy, chemotherapy, radiotherapy (as includes but not limited to weak, anaemia, emaciation etc.), chronic poisoning by salicylic acid salt etc.Referring to as Merck Manual, 12-17 version, Merck ﹠amp; Company, Rahway, NJ (1972,1977,1982,1987,1992,1999), Pharmacotherapy Handbook, Wells etc. compile the 2nd edition, Appleton and Lange, Stamford, Conn. (1998,2000) is incorporated herein by reference separately fully.
The present invention also provides a kind of and has been used for regulating or the treatment cell, tissue, organ, the method of at least a cardiovascular disorder among animal or the patient, described cardiovascular disorder includes but not limited at least a heart stun syndrome (cardiac stun syndrome), myocardial infarction, congestive heart failure, apoplexy, ischemic stroke, hemorrhage, acute coronary syndrome, arteriosclerosis, atherosclerosis, restenosis, diabetes atheromatosis (diabetic ateroscleroticdisease), hypertension, arterial hypertension, renovascular hypertension, faint, shock, cardiovascular systems syphilis, in heart failure, lung (former) property heart trouble, primary pulmonary hypertension, irregular pulse, the atrium ectopic beats, auricular flutter, atrial fibrillation (persistence or sudden), postperfusion syndrome, the cardiopulmonary bypass inflammatory reaction, irregularity or multifocal atrial tachycardia, regular narrow QRS tachycardia, specific arrhythmia, ventricular fibrillation, Xi Shi restraints irregular pulse (His bundle arrythmias), atrioventricular block, bundle branch block, myocardial ischaemia venereal disease disease, coronary artery disease, stenocardia, myocardial infarction, myocardosis, DCMP, restrictive cardiomyopathy, valvular heart disease, endocarditis, pericardial disease, cardiac tumor, aorta and peripheral aneurysm, the aorta wall interlayer forms, the aorta inflammation, aorta abdominalis and branch's obturation thereof, the peripheral blood vessel illness, obliterative arteriopathy disease, the atherosis disease of peripheral arterial (peripheral atherlosclerotic disease), thromboangiitis obliterans, function peripheral arterial illness, Raynaud's phenomenon and disease, acrocyanosis, erythromelalgia, venous disease, thrombophlebitis, cirso-, arterio venous fistula, lymphedema, lipedema, unstable angina, reperfusion injury, syndrome behind the pump (post pumpsyndrome), ischemia reperfusion injury etc.Such method can comprise randomly that the composition that comprises at least a anti-IL-6 antibodies of using significant quantity or pharmaceutical composition are in cell, tissue, organ, animal or the patient of the such adjusting of needs, processing or treatment.
The present invention also provides a kind of and has been used for regulating or the treatment cell, tissue, organ, the method of the relevant transmissible disease of at least a IL-6 among animal or the patient, the relevant transmissible disease of described IL-6 includes but not limited at least a: acute or chronic bacterial infection, acute or chronic parasitism or infectious process, comprise bacterium, virus and fungi infestation, HTV infection/HIV DPN, meningitis, hepatitis is (as the first type, B-mode or third type etc.), septic arthritis, peritonitis, pneumonia, epiglottitis, colon bacillus 0157: h7, hemolytic uremic syndrome/thrombolysis thrombopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, suis myositis, gas gangrene, Mycobacterium tuberculosis, mycobacterium avium born of the same parents endophyte, pneumocystis carinii pneumonia, inflammatory pelvic disease, testitis/epididymitis, Legionnella, Lyme disease, influenza A, Epstein-Barr virus, the hemophagocytic syndrome that virus is relevant, viral encephalitis/aseptic meningitis etc.
The present invention also provides a kind of and has been used for regulating or the treatment cell, tissue, organ, the method of the relevant malignant diseases of at least a IL-6 among animal or the patient, the relevant malignant diseases of described IL-6 include but not limited at least a: leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphoblastic leukemia, the B-cell, T-cell or FAB ALL, acute myelogenous leukemia (AML), acute myeloid leukaemia, chronic myelocytic leukemia (CML), lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hokdkin disease, malignant lymphoma, non_hodgkin lymphoma, Burkitt lymphoma, multiple myeloma, Kaposi, colorectal carcinoma, carcinoma of the pancreas, nasopharyngeal carcinoma, malignant histocytosis, paraneoplastic syndrome/pernicious hypercalcemia (the special property sent out) syndrome, solid tumor, bladder cancer, mammary cancer, colorectal cancer, carcinoma of endometrium, cancer, the neck cancer, the hereditary nonpolyposis cancer, He Jiejin (family name) lymphoma, liver cancer, lung cancer, nonsmall-cell lung cancer, ovarian cancer, the pancreas cancer, prostate cancer, renal cell carcinoma, carcinoma of testis, gland cancer, sarcoma, malignant melanoma, vascular tumor, metastatic disease, Cancer-Related bone resorption, Cancer-Related ostalgia etc.
The present invention also provides a kind of method that is used for regulating or treating the relevant sacred disease of at least a IL-6 of cell, tissue, organ, animal or patient, the relevant sacred disease of described IL-6 includes but not limited at least a: neurodegenerative disease, multiple sclerosis, migraine, AIDS chronic brain syndrome, demyelinating diseases, for example multiple sclerosis and acute transverse myelitis; Pyramidal tract outer and cerebellum illness, for example cortex spinal cord system damage; The basal ganglion illness; Hyperkinetic dyskinesia, for example huntington's chorea and senile chorea; Drug-induced dyskinesia is for example for blocking those illnesss that medicine brought out of CNS Dopamine Receptors; Hypokinetic dyskinesia, for example Parkinson's disease; Carrying out property pronucleus paralysis (Progressive supranucleo Palsy); The cerebellum structural damage; Spinocerebellar degeneration, spinal ataxia for example, Freed rely uncommon (family name) ataxia, cerebellar cortex sex change, multiple system degeneration (Mencel, Dejerine-Thomas, Shi-Drager and Machado-Joseph); Systemic disorders (the not plain nurse of thunder (family name) disease, abetalipoprotemia, ataxia, telangiectasis and plastosome multisystem illness); Demyelination nuclear illness (demyelinating coredisorders), multiple sclerosis for example, acute transverse myelitis; And moving cell illness neurotic atrophy (anterior horn cell sex change, for example amyotrophic lateral sclerosis (spinal cord) lateral sclerosis, infantile spinal muscular atrophy and JSMA) for example; Alzheimer; Mongolism among the middle-aged people; Diffuse Lewy body disease; Lewy build senile dementia; Wernicke-Korsakoff syndrome; Dipsorrhexia; Creutzfeldt-Jakob disease; Subacute sclerosing panencephalitis, Hallerrorden-Spatz disease; Dementia pugilistica; Neurotrauma (as Spinal injury, brain injury, cerebral concussion, repeated cerebral concussion); Pain; Inflammatory pain; Autism; Dysthymia disorders; Apoplexy; Cognitive disorder; Epilepsy etc.Such method can randomly comprise use comprising of significant quantity at least a TNF antibody or the composition of specific part or variant or pharmaceutical composition in cell, tissue, organ, animal or the patient of the such adjusting of needs, processing or treatment.Referring to as the Merck Manual, the 16th edition, Merck ﹠amp; Company, Rahway, NJ (1992).
The present invention also provides a kind of method that is used for regulating or treat at least a IL-6 of cell, tissue, organ, animal or patient relevant wound, wound or tissue injury or relevant chronic disease, wound, wound or tissue injury that described IL-6 is relevant or relevant chronic disease include but not limited to, and be at least a: with the relevant somatic damage or the wound of oral surgery of the application and the use of the insertion that comprises periodontal surgical operation, exodontia, endodontic treatment, dental implants, prosthodontics; Or wherein wound for be selected from aseptic wound, contusion, incised wound, lacerated wound, non perforating wound, open wound, penetrating wound, perforating wound, stab, infected wound, infarction and subcutaneous wound; Or wherein wound for be selected from ischemic ulcer, ischemic ulcer, fistula, seriously bite, thermal burn and donor site wound; Or wherein wound is the relevant wound of aphtha wound, traumatic wounds or bleb.
Wound and/or ulcer see on skin protuberance or the mucomembranous surface usually or are the result of organ infarction (" apoplexy ").Wound can be the result of soft tissue defects or infringement or potential illness.In the context of the invention, term " skin " relates to the outermost surface of the health of the animal that comprises the people, and comprises int or almost int skin and impaired skin surface.Term " mucous membrane " relates to the mucous membrane of int or impaired animal such as the people, can be oral mucosa, buccal mucosa, ear mucous membrane, nasal mucosa, lung mucous membrane, eye mucosa, gastrointestinal mucosa, vaginal mucosa or mucous membrane of rectum.
In the context of the invention, term " wound " refers to have the somatic damage of weave construction normal integrity destructive.This term also is intended to comprise term " sore ", " infringement ", " necrosis " and " ulcer ".Usually, term " sore " is the generic term of nearly all skin or mucosal injury, and term " ulcer " is SOL or the pouch that produces from the organ or tissue surface that necrotic tissue comes off.Infringement is relevant with any tissue defect usually.Downright bad and infection, damage, inflammation or the thanatogenic tissue that infarction produced are relevant.
The term " wound " that is used for the context of the invention refers to any wound (referring to following classification to wound) and be in any specified phase in healing process, be included in any healing before beginning stage or even resemble surgical incision (prophylactic treatment) stage before producing specific wound.The examples of trauma that can be prevented and/or treated according to the present invention is, as aseptic wound, contusion, incised wound, lacerated wound, non perforating wound (promptly wherein skin is not damaged, but its wound of structural damage evil down), open wound, penetrating wound, perforating wound, stab, infected wound, subcutaneous wound etc.The example of sore is bedsore (bed sores), aphtha, chrome ulcer, cold sore, bedsore (pressure sores) etc.The example of ulcer is, as peptide ulceration, duodenal ulcer, stomach ulcer, gout ulcer, diabetic ulcer, hypertensive ischemic ulcer, stasis ulcer, ulcus cruris (varicose ulcer), sublingual ulcer, submucous ulcer, symptomatic ulcer, trophic ulcer, Malabar ulcer with as the caused venereal ulcer of gonorrhoea (comprising urethritis, endocervicitis and rectitis).The illness relevant with wound or sore that can successfully treat according to the present invention is burn, anthrax, tetanus, gas gangrene, scarlet fever, erysipelas, Sycosis vulgaris, folliculitis, impetigo contagiosa or BI etc.Between the use of term " wound " and " ulcer " and " wound " and " sore ", to a certain degree overlapping arranged usually, in addition, these terms can use arbitrarily usually.Therefore, as mentioned above, in the context of the invention, term " wound " comprises term " ulcer ", " infringement ", " sore " and " infarction ", and unless otherwise stated, otherwise these terms can indistinguishably use.
The kind of the wound of being treated according to the present invention also comprises (i) general wound, for example surgery, traumatic, infectious, ischemic, heat, chemistry and bleb wound; The (ii) distinctive wound in oral cavity, exodontia back wound for example, dental pulp wound especially with purulence born of the same parents and abscess, bacterium, virus or autoimmunization source ulcer and damage, machinery, chemistry, heat, the infection wound relevant with liver moss sample wound treatment; Bleb ulcer, aphthous stomatitis, acute necrotizing ulcerative gingivitis and burning mouth syndrome are concrete examples; (iii) skin wound, bites and surgical incision at for example vegetation, burn (as chemical burn, thermal burn), damage (bacterium, virus, autoimmunization).Another kind of wound mode classification for (i) because the little tissue loss due to surgical incision, less galling and less the biting, or (ii) tangible tissue loss.One group of back comprises ischemic ulcer, bedsore, fistula, lacerated wound, seriously bites, thermal burn and donor site wound (in soft tissue and sclerous tissues) and infarction.
Other important wounds relevant with the present invention for such as ischemic ulcer, bedsore, fistula, seriously bite, the wound of thermal burn and donor site wound.Ischemic ulcer and bedsore be only heal usually wound and especially under these circumstances very slowly, improvement and fast agglutination that yes is very important for the patient.In addition, when healing was improved and is quick more, relevant cost with the patient treatment of suffering from such wound will significantly reduce.
The donor site wound for for example with remove sclerous tissues from a health part to the relevant wound that is occurred of health another part, as with transplant relevant wound.Such wound that operation produced is pain very, and therefore the healing that improves is a most worthy.Term " skin " uses with implication very widely, comprise skin epidermal area and-therein under more or less impaired those situations of skin surface-also comprise the skin corium of skin.Except that stratum corneum, the epidermal area of skin is outer (epithelium) layer, and darker skin connective tissue layer is called corium.
The invention provides a kind of be used for regulating or treating cell, tissue, organ, animal or patient's osteoarthritis, systemic lupus erythematosus, lupus erythematosus,cutaneous, lupus nephritis, type ii diabetes and chronic obstructive pulmonary disease, and the method for the relevant other diseases of IL-6 as listed above, the relevant other diseases of described IL-6 includes but not limited to, at least a immune correlated disease, cardiovascular disorder, transmissible disease, malignant diseases and/or sacred disease.Such method can comprise randomly that the composition that comprises at least a anti-IL-6 antibodies of using significant quantity or pharmaceutical composition are in cell, tissue, organ, animal or the patient of the such adjusting of needs, processing or treatment.
Any method of the present invention can comprise that the composition that comprises at least a anti-IL-6 antibodies of using significant quantity or pharmaceutical composition are in cell, tissue, organ, animal or the patient of the such adjusting of needs, processing or treatment.Such method can randomly further comprise administration or combination therapy when being used for the treatment of such disease or illness, wherein said at least a anti-IL-6 antibodies, the using of its specific part or variant further is included in before it uses, simultaneously and/or use afterwards be selected from least a TNF antagonist (as but be not limited to TNF chemistry or protein antagonist, TNF mono-clonal or polyclonal antibody or fragment, soluble TNF acceptor is (as p55, p70 or p85) or fragment, their fusion polypeptide, or small molecules TNF antagonist, as conjugated protein I of TNF or II (TBP-I or TBP-II), nerelimonmab, infliximab, etanercept, CDP-571, CDP-870, Afelimomab, Lenercept etc.), antirheumatic is (as methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, disodium aurothiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), muscle relaxant, narcotic, NSAID (non-steroidal anti-inflammatory drug) (NSAID), anodyne, narcotic, tranquilizer, local anesthetic, neuromuscular blocking agents, biocide is (as glucosaminide, antifungal drug, antiparasitic, antiviral drug, carbon (mixing) penem, cynnematin, fluoroquinolone, macrolide, penicillin, sulfanilamide (SN), tsiklomitsin, other biocides), antipsoriatic, corticosteroid, anabolic steroid, the medicine that diabetes are relevant, mineral substance, nutrition, thyroid, VITAMIN, the hormone that calcium is relevant, diarrhea, cough medicine, antiemetic, anti-ulcerative drug, laxative, anti-coagulant, erythropoietin (as the epoetin), filgrastim is (as G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunization, immunoglobulin (Ig), immunosuppressor is (as basiliximab, ciclosporin, reach (gram) pearl monoclonal antibody), tethelin, the hormone replacement medicine, estrogenic agents, iridodilator, cycloplegic, alkylating agent, antimetabolite, mitotic inhibitor, radioprotector, thymoleptic, antimaniacal drugs, antipsychotic drug, antianxiety agent, soporific, sympathomimetic, stimulant, E2020, tacrine, asthma drug, beta-agonists, the steroid class that sucks, the leukotrienes inhibitor, methyl xanthine, Cromoglycic Acid, suprarenin or analogue, α streptodornase (Pulmozyme), cytokine or cytokine antagonist at least a.The dosage that is fit to is well-known in the art.Referring to as Wells etc., compile Pharmacotherapy Handbook, the 2nd edition, Appleton andLange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon PocketPharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000); Nursing 2001 Handbook of Drugs, the 21st edition, SpringhouseCorp., Springhouse, PA, 2001; Health Professional ' s Drug Guide 2001, editor, Shannon, Wilson, Stang, Prentice-Hall, tic, Upper Saddle River, NJ., every piece of document all is incorporated herein by reference fully at this.
The TNF antagonist that is suitable for the present composition, combination therapy, administration simultaneously, device and/or method (further comprising at least a antibody of the present invention, its specific part and variant) includes but not limited to that anti-TNF antibodies (as the TNF antagonist of at least a as above definition), its Fab and specificity are in conjunction with the acceptor molecule of TNF; Stop and/or suppress compound that TNF is synthetic, TNF discharges or it acts on target cell, for example thalidomide, tenidap, phosphodiesterase inhibitor (as pentoxifylline and rolipram), A2b adenosine receptor agonist and A2b adenosine receptor toughener; Stop and/or suppress the compound of TNF receptor signal conduction, for example mitogen activated protein (MAP) kinase inhibitor; The compound of blocking-up and/or inhibition film TNF cutting, for example inhibitors of metalloproteinase; Blocking-up and/or the active compound of inhibition TNF, for example vasotonia speed saccharase (ACE) inhibitor (as captopril); And blocking-up and/or inhibition TNF generation and/or synthetic compound, for example map kinase inhibitor.
As used herein " tnf antibody ", " TNF antibody ", " TNF Alpha antibodies " or fragment etc. reduce, blocking-up, suppress, eliminate or disturb that TNF α is external, original position and/or activity in vivo preferably.For example, TNF people's antibody of the present invention of Shi Heing can in conjunction with TNF α and comprise anti-TNF antibodies, its Fab and specified mutant or its specificity in conjunction with the structural domain of TNF α.TNF antibody that is fit to or fragment also can reduce, block, eliminate, disturb, stop and/or suppress TNF RNA, DNA or albumen is synthetic, and TNF discharges, the conduction of TNF receptor signal, and film TNF cutting, the TNF activity, TNF produces and/or is synthetic.
The example of TNF antibody or antagonist is chimeric antibody cA2.The example that can be used for the mono-clonal anti-TNF antibodies among the present invention in addition described in the art is (referring to as the U.S. patent No. 5,231,024;
Figure A200680024037D0071143332QIETU
, A. etc., Cytokine 2 (3): 162-169 (1990); U.S. application number 07/943,852 (on September 11st, 1992 submitted to); Rathjen etc., international publication number WO91/02078 (on February 21st, 1991 announced); Rubin etc., EPO patent publication No. 0218868 (on April 22nd, 1987 announced); Yone etc., EPO patent publication No. 0 288 088 (on October 26th, 1988); Liang, etc., Biochem.Biophys.Res.Comm.237:847-854 (1986); Meager, etc., Hybridoma 6:305-311 (1987); Fendly etc., Hybridoma 6:359-369 (1987); Bringman, etc., Hybridoma 6:489-507 (1987); And Hirai, etc., J.Immunol.Meth.96:57-62 (1987).
The TNF acceptor molecule
The preferred TNF acceptor molecule that is used for the present invention be those with high-affinity in conjunction with TNF α and randomly have reduced immunogenicity acceptor molecule (referring to as Feldmann etc., international publication number WO 92/07076 (on April 30th, 1992 announced); Schall etc., Cell67:361-370 (1990); With Loetscher etc., Cell 67:351-359 (1990), these documents are incorporated herein by reference fully at this).Particularly, use 55kDa (p55TNF-R) and 75kDa (p75 TNF-R) TNF cell surface receptor in the present invention.The clipped form (referring to as Corcoran etc., Eur.J.Biochem.223:831-840 (1994)) that comprises these acceptors of receptor extracellular domain (ECD) or its funtion part also is used for the present invention.In urine and serum, detected the TNF acceptor clipped form that comprises ECD, be respectively 30kDa and 40kDa TNF α and suppress conjugated protein (Engelmann, H. etc., J.Biol.Chem.265:1531-1536 (1990)).TNF acceptor multimeric molecule and TNF immunity receptor fusion molecule and derivative thereof and fragment or part are the examples that is used for the extra TNF acceptor of the inventive method and composition.
Be used for the whole or funtion part that TNF acceptor multimeric molecule of the present invention comprises the ECD of two or more TNF acceptors that connect by one or more peptide linkers or such as the non-peptide linkers of other of polyoxyethylene glycol (PEG).An example of such TNF immunity receptor fusion molecule is TNF acceptor/IgG fusion rotein.TNF immunity receptor fusion molecule and the method (Lesslauer etc., the Eur.J.Immunol.27:2883-2886 (1991) that produce it had been described in this area already; Ashkenazi etc., Proc.Natl.Acad.Sci.USA 88:10535-10539 (1991); Peppel etc., J.Exp.Med.774:1483-1489 (1991); KoUs etc., Proc.Natl.Acad.Sci.USA 97:215-219 (1994); Butler etc., Cytokine 6 (6): 616-623 (1994); Baker etc., Eur.J.Immunol.24:2040-2048 (1994); Beutler etc., the U.S. patent No. 5,447,851; With U.S. application number 08/442,133 (submit to May 16 nineteen ninety-five), every piece of document all is incorporated herein by reference fully at this).The method that is used to produce the immunity receptor fusion molecule is also shown in Capon etc., the U.S. patent No. 5,116,964; Capon etc., the U.S. patent No. 5,225,538 and Capon etc., Nature 357:525-531 (1989), these documents are incorporated herein by reference fully at this.
Cytokine comprises any known cytokine.Referring to as CopewithCytokines.com.Cytokine antagonist includes but not limited to any antibody, fragment or stand-in, any soluble receptors, fragment or stand-in, any small molecules antagonist or their any combination.
Treatment is handled
Any method of the present invention can comprise the method for the illness that is used for the treatment of IL-6 mediation, comprises that the composition that comprises at least a anti-IL-6 antibodies of using significant quantity or pharmaceutical composition are in cell, tissue, organ, animal or the patient of the such adjusting of needs, processing or treatment.Such method can randomly further comprise administration or combination therapy when being used for the treatment of such disease or illness, wherein said at least a anti-IL-6 antibodies, the using of its specific part or variant further is included in before it uses, simultaneously and/or use afterwards and be selected from anti-infective, cardiovascular (CV) system medicine, central nervous system (CNS) medicine, autonomous nervous system (ANS) medicine, the respiratory tract medicine, stomach and intestine (GI) road medicine, hormone drug, the medicine that is used for water or electrolyte balance, hematologic drug, antitumor agent, immunomodulator, medicament for the eyes, ear medicine or nose medicine, topical drug, nutritional drugs etc., at least a TNF antagonist (as but be not limited to TNF antibody or fragment, soluble TNF acceptor or fragment, their fusion rotein or small molecules TNF antagonist), antirheumatic is (as methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, disodium aurothiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), muscle relaxant, narcotic, NSAID (non-steroidal anti-inflammatory drug) (NSAID), anodyne, narcotic, tranquilizer, local anesthetic, neuromuscular blocking agents, biocide is (as glucosaminide, antifungal drug, antiparasitic, antiviral drug, carbon (mixing) penem, cynnematin, fluoroquinolone, macrolide, penicillin, sulfanilamide (SN), tsiklomitsin, other biocides), antipsoriatic, corticosteroid, anabolic steroid, the medicine that diabetes are relevant, mineral substance, nutrition, thyroid, VITAMIN, the hormone that calcium is relevant, diarrhea, cough medicine, antiemetic, anti-ulcerative drug, laxative, anti-coagulant, erythropoietin (as the epoetin), filgrastim is (as G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunization, immunoglobulin (Ig), immunosuppressor is (as basiliximab, ciclosporin, reach (gram) pearl monoclonal antibody), tethelin, the hormone replacement medicine, estrogenic agents, iridodilator, cycloplegic, alkylating agent, antimetabolite, mitotic inhibitor, radioprotector, thymoleptic, antimaniacal drugs, antipsychotic drug, antianxiety agent, soporific, sympathomimetic, stimulant, E2020, tacrine, asthma drug, beta-agonists, the steroid class that sucks, the leukotrienes inhibitor, methyl xanthine, Cromoglycic Acid, suprarenin or analogue, α streptodornase (Pulmozyme), cytokine or cytokine antagonist at least a.Such medicine is that this area is well-known, and composition, indication, dosage and the drug regimen that comprises every kind of medicine all listed in this (referring to as Nursing2001 Handbook of Drugs, the 21st edition, Springhouse Corp., Springhouse, PA, 2001; Health Professional ' s Drug Guide 2001, editor, Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ; PharmcotherapyHandbook, Wells etc., compile Appleton; Lange, Stamford, CT, each comfortable this is incorporated herein by reference fully).
Typically, carry out the treatment of pathological condition by at least a anti-IL-6 antibodies composition of using significant quantity or dosage, the specific activity of the promoting agent that depends in the composition to be contained, significant quantity or dosage add up on average at least about at least a anti-IL-6 antibodies of 0.01-500 milligram/kilogram patient/dosage, and preferably, at least about 0.1-100 milligram antibody/kilogram patient/dosage or repeatedly use.Perhaps, effectively serum-concentration can comprise that 1-5000 μ g/ml serum-concentration/single or multiple uses.The dosage that is fit to is known to the medical science practitioner, and should depend on concrete disease condition certainly, the specific activity of the composition used and the concrete treatment that the patient experienced.In some cases,, may need to provide repetitive administration, promptly repeat using separately of the concrete dosage of monitoring or measuring, wherein repeat to use separately till per daily dose that reaches expectation or effect for reaching the desired therapeutic amount.
Preferred dosage can randomly comprise about 0.1-99 and/or 100-500mg/kg/ administration, or its any scope, value or mark, or reach the serum-concentration that about 0.1-5000 μ g/ml serum-concentration/single or multiple is used, or its any scope, value or mark.The dosage range of preferred anti-IL-6 antibodies of the present invention is the about 1mg of per kilogram weight in patients extremely about 3, about 6 or about 12mg.
Perhaps, dosage can according to such as the drug effect characteristic of concrete medicament with and administering mode and approach; Receptor's age, health and body weight; The nature and extent of symptom, the type of combination therapy, the known facts of therapeutic frequency and desired effects and changing.The dosage of activeconstituents generally can be the about 0.1-100mg of per kilogram of body weight.Be generally 0.1-50, and be preferably the 0.1-10mg/kg/ administration or be sustained release form with effective acquisition expected result.
As nonrestrictive example, utilize single, infusion or multidose, at least one sky at 1-40 days, or at least one week in 1-52 week, or at least one year at 1-20, or their any combination, provide every day about 0.1-100mg/kg or its any scope, value or at least a antibody of the present invention of fractional once or cycle dose as human or animal's treatment.
The formulation that is suitable for vivo medicine-feeding (composition) of per unit or container contains the activeconstituents of the about 500mg of 0.001mg-that has an appointment usually.In these pharmaceutical compositions, activeconstituents usually with based on the composition gross weight by weight the amount of about 0.5-99.999% exist.
For administered parenterally, antibody can combine or provide separately and be formulated as solution, suspension, emulsion, particle, powder or lyophilized powder with pharmaceutically acceptable parenteral carrier.The example of such carrier is water, salt solution, Ringer's solution, glucose solution and about 1-10% human serum albumin.Also can use for example fixed oil of liposome and nonaqueous carrier.Carrier or lyophilized powder can contain the additive that keeps isotonicity (as sodium-chlor, mannitol) and chemical stability (as buffer reagent and sanitas).By known or suitable technology sterilization prescription.
The pharmaceutical carrier that is fit to is described in nearest one edition this field canonical reference textbook Remington ' s Pharmaceutical Sciences, among the A.Osol.
The selectivity administration
According to the present invention, can use multiple known and the exploitation mode use pharmacy effective dose according at least a anti-IL-6 antibodies of the present invention.When having used in the following description, can use other administering modes that have suitable result according to the present invention through the lung administration.Utilization is suitable for multiple apparatus and method any of described here or other administering modes known in the art of inhalation, and IL-6 antibody of the present invention can be in the carrier, as solution, emulsion, colloid or suspension, or sends as dry powder.
Parenteral prescription and administration
The prescription that is used for administered parenterally can contain vehicle-sterilized water commonly used or salt solution, polyalkenyl ethylene glycol, for example polyoxyethylene glycol, vegetables oil, hydrogenated naphthalene etc.Can be according to currently known methods by using the moisture or oleaginous suspension of suitable emulsifying agent or moistening agent and suspending agent preparation for injection.Injection reagent can be nontoxic, non-oral thinner, for example the aqueous solution, aseptic injectable solution or be in suspension in the solvent.As available carrier or solvent, provide water, Ringer's solution, etc. infiltration salt solution etc.; Can use aseptic fixed oil as common solvent or suspending agent.For these purposes, can use the fixed oil or the lipid acid of any kind, comprise natural or synthetic or semisynthetic fatty oil or lipid acid; Natural or synthetic or semisynthetic glycerine list-or two-or three esters.Administered parenterally is known in the art, includes but not limited to conventional injecting method, as the U.S. patent No. 5,851, the gas-pressured needled-less head injection device of describing in 198 and as the U.S. patent No. 5, the laser beam perforation device of describing in 839,446 is incorporated herein by reference fully at this.
Selectivity is sent
The invention further relates to and pass through parenteral, subcutaneous, intramuscular, intravenously, intraarticular, in the segmental bronchus, in the abdomen, in the capsule, in the cartilage, in the chamber, in the body cavity, in the cerebellum, Intraventricular, colonic, in the uterine cervix, in the stomach, in the liver, in the cardiac muscle, in the bone, in the pelvis, in the pericardium, intraperitoneal, in the pleura, in the prostate gland, in the lung, internal rectum, in the kidney, in the retina, in the backbone, in the synovial membrane, in the thoracic cavity, intrauterine, intravesical, in the damaging part, inject, vagina, rectum, the oral cavity contains clothes, the hypogloeeis, use at least a anti-IL-6 antibodies in the nose or through the skin method.Can prepare at least a anti-IL-6 antibodies composition, be used for parenteral (subcutaneous, intramuscular or intravenously) or any other administration, particularly the form with liquor or suspension exists; Be used for vagina or rectal administration particularly exists with semi-solid form, such as but not limited to emulsifiable paste and suppository; Be used for the oral cavity and contain clothes or sublingual administration, such as but not limited to existing with tablet or capsule form; Or be used for intranasal administration, such as but not limited to, powder, nasal drop or aerosol or some reagent form; Or be used for percutaneous dosing, such as but not limited to gel, ointment, lotion, suspension or have such as dimethyl sulfoxide (DMSO) to change skin texture or to increase the chemical intensifier (Junginger of transdermal patch Chinese traditional medicine concentration, Deng, at ＂ Drug Permeation Enhancement; Hsieh among the ＂, D.S., compile, pp.59-90 (Marcel Dekker, Inc.New York 1994 is incorporated herein by reference fully at this), or have and can use the oxygenant (WO 98/53847) of prescription on skin that contains albumen and peptide, or apply electric field to produce of short duration transportation path, electroporation for example, or increase the percutaneous movability of charged medicine, for example iontophoresis, or apply ultrasonic wave, phonophoresis (the U.S. patent No. 4,309,989 and 4 for example, 767,402) patch delivery system (above publication and patent are incorporated herein by reference fully at this).
Through lung/intranasal administration
For through the lung administration, preferably, at least a anti-IL-6 antibodies composition is sent with the granularity of subordinate's air flue of arriving lung or sinus effectively.According to the present invention, can send at least a anti-IL-6 antibodies by multiple be used for installing in suction known in the art by sucking the administering therapeutic agent or the nose any.These can deposit the device of aerosol formulations in patient's sinus chamber or groove and comprise metered dose inhaler, spraying gun, dry powder producer, atomizer etc.Other are suitable for antibody also is known in the art through the device of lung or intranasal administration directly.All such devices all can use and be suitable for the prescription that the administration distribution is in the antibody in the aerosol.Such aerosol can be made up of solution (moisture and non-water) or solid particulate.
Metered dose inhaler as
Figure A200680024037D00761
Metered dose inhaler has used propellent usually and has needed to start (referring to as WO 94/16970, WO 98/35888) during sucking.Diskus such as Turbuhaler TM(Astra),
Figure A200680024037D00762
(Glaxo),
Figure A200680024037D00763
(Glaxo), Spiros TMThe device that sucker (Dura), Inhale Therapeutics are sold and
Figure A200680024037D00764
Diskus (Fisons) has used mixed powder (US 4668218 Astra, the EP 237507Astra of respiration drive, WO 97/25086 Glaxo, WO 94/08552 Dura, US 5458135 Inhale, WO94/06498 Fisons is incorporated herein by reference fully at this).Spraying gun such as AERx TMAradigm,
Figure A200680024037D00765
Spraying gun (Mallinckrodt) and
Figure A200680024037D00766
Spraying gun (MarquestMedical Products) (US 5404871 Aradigm, WO 97/22376), above-mentioned document is incorporated herein by reference fully at this, has produced aerosol by solution, and metered dose inhaler, Diskus etc. has then produced the particulate aerosol.The specific examples of these commercially available suction apparatus is intended to represent the specific device that is suitable for the present invention's practice, and is not intended to limit the scope of the invention.
Preferably, send the composition that comprises at least a anti-IL-6 antibodies by Diskus or atomizer.For using at least a antibody of the present invention, suction apparatus has several needed characteristics.For example, be reliably, can repeat by the suction apparatus delivery advantage and accurately.Suction apparatus can randomly be sent dry particles, as less than about 10 μ m, and preferably about 1-5 μ m, very respirability.
Use the IL-6 antibody compositions as sprays
Can produce the sprays that comprises the IL-6 antibody compositions by nozzle by the suspension or the solution of at least a anti-IL-6 antibodies that pressurizes.Can select jet size and profile, institute's applied pressure and liquid feeding speed to obtain the work output and the granularity of expectation.Can for example produce the electron spray(ES) agent by electric field with kapillary or nozzle material-feeding pipe jointing.Advantageously, the particle of at least a anti-IL-6 antibodies composition of sending by atomizer has the granularity less than about 10 μ m, preferably, in about 5 mu m ranges of about 1 μ m-, and most preferably, is the about 3 μ m of about 2 μ m-.
Be applicable to that the prescription of at least a anti-IL-6 antibodies composition of atomizer is usually included at least a anti-IL-6 antibodies composition/ml soln or the mg/gm of the about 100mg of about 0.1mg-, or the antibody compositions that exists with aqueous solution form under any scope wherein, value or the fractional concentration.Prescription can comprise such as vehicle, buffer reagent, isotonic agent, sanitas, tensio-active agent and the reagent of zinc preferably.Prescription also can comprise vehicle or be used for the reagent of stabilization of antibodies composition, for example buffer reagent, reductive agent, filling albumen (bulk protein) or carbohydrate.The filling albumen that is used to prepare antibody compositions comprises albumin, protamine etc.The representational carbohydrate that is used to prepare antibody compositions comprises sucrose, mannitol, lactose, trehalose, glucose etc.The antibody compositions prescription also can comprise tensio-active agent, and it can reduce or stop because the antibody compositions of the caused spatial induction of solution atomization gathering in aerosol forms.Can use the tensio-active agent of multiple routine, for example polyoxyethylene fatty acid ester class and alcohols, and polyoxyethylene sorbitol fatty acid ester.The amount of tensio-active agent is generally the 0.001-14% of prescription by weight.For purposes of the invention, particularly preferred tensio-active agent is polyoxyethylene sorbitan monooleate, polysorbate80, polysorbate20 etc.Known in the art being used for also can be included in prescription such as the additional agents of the protein prescription of IL-6 antibody or specific part or variant.
Use the IL-6 antibody compositions by spraying gun
Can be by using antibody compositions of the present invention such as the spraying gun of blast atomizer or ultrasonic atomizer.Typically, in blast atomizer, use compressed air source to produce high-velocity jets by nozzle.When the gas jetting nozzle, produced low pressure area, extract antibody compositions solution by the kapillary that links to each other with liquid vessel.When it leaves, be cut into unsettled filament and droplet from liquid stream capillaceous, produce aerosol.Can use multiple profile, flow velocity and inducer type to obtain the performance of expectation by given blast atomizer.In ultrasonic atomizer, use PZT (piezoelectric transducer) that high-frequency electrical energy is used to produce vibrational energy, mechanical energy usually.This energy directly or by connecting coupled fluid is sent to the antibody compositions prescription, produces the aerosol that comprises antibody compositions.Advantageously, the antibody compositions particle of sending by spraying gun has the granularity less than about 10 μ m, preferably in about 5 mu m ranges of about 1 μ m-, and most preferably, is the about 3 μ m of about 2 μ m-.
Be applicable to that the prescription of at least a anti-IL-6 antibodies of spraying gun (spraying or ultrasonic atomizer) generally includes the proteic concentration of at least a anti-IL-6 antibodies of the about 100mg of the about 0.1mg-of every ml soln.Prescription can comprise such as vehicle, buffer reagent, isotonic agent, sanitas, tensio-active agent and the reagent of zinc preferably.Prescription also can comprise vehicle or be used to stablize the reagent of at least a anti-IL-6 antibodies composition, for example buffer reagent, reductive agent, filling albumen or carbohydrate.The filling albumen that is used to prepare at least a anti-IL-6 antibodies composition comprises albumin, protamine etc.The representational carbohydrate that is used to prepare at least a anti-IL-6 antibodies comprises sucrose, mannitol, lactose, trehalose, glucose etc.At least a anti-IL-6 antibodies prescription also can comprise tensio-active agent, and it can reduce or stop because at least a anti-IL-6 antibodies gathering of the caused spatial induction of solution atomization in aerosol forms.Can use the tensio-active agent of multiple routine, for example polyoxyethylene fatty acid ester class and alcohols, and polyoxyethylene sorbitol fatty acid ester.The amount of tensio-active agent is generally the 0.001-4% of prescription by weight.For purposes of the invention, particularly preferred tensio-active agent is polyoxyethylene sorbitan monooleate, polysorbate80, polysorbate20 etc.Known in the art being used for also can be included in prescription such as the additional agents of the protein prescription of antibody protein.
Use the IL-6 antibody compositions by metered dose inhaler
In metered dose inhaler (MDI), propellent, at least a anti-IL-6 antibodies and any vehicle or other additive-package are contained in the metal tin as the mixture that comprises liquified compressed gas.The startup metering valve has discharged the mixture as aerosol, preferably contains the particle less than the size range of about 10 μ m, is preferably about the about 5 μ m of 1 μ m-, and is most preferably the about 3 μ m of about 2 μ m-.Can obtain the aerosol granularity of expectation by the prescription that uses the antibody compositions that produces by multiple method known to those skilled in the art, described method comprises jet grinding, spraying drying, stagnation point condensation etc.Preferred metered dose inhaler is included as those gaugers 3M or Glaxo manufacturing and that use the hydrogen fluorocarbon propellant.The prescription that is used at least a anti-IL-6 antibodies of metered dose inhaler device generally includes as the finely divided powder that contains at least a anti-IL-6 antibodies that is in the suspended matter of anhydrous medium, for example, is suspended in the propellent by means of tensio-active agent.Propellent can be any conventional substances that is used for this purposes, for example chlorine fluorocarbon, hydrogen chlorine fluorocarbon, hydrogen fluorocarbon or hydrocarbon polymer, comprise trichlorofluoromethane, Refrigerant 12, Dichlorotetrafluoromethane and 1,1,1,2-tetrafluoroethylene, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227) etc.Preferably, propellent is the hydrogen fluorocarbon.Can select tensio-active agent to stablize as at least a anti-IL-6 antibodies that is in the suspended matter in the propellent, so that activeconstituents avoids chemical degradation etc.The tensio-active agent that is fit to comprises sorbitanic trioleate, soybean phospholipid, oleic acid etc.In some cases, it is preferred using the solution aerosol such as the alcoholic acid solvent.The additional agents that is used for protein prescription known in the art also can be included in prescription.Those skilled in the art agree approval and use at least a anti-IL-6 antibodies composition by the device that does not have at this to describe through lung and realize method of the present invention.
Formula of oral and administration
Be used for oral prescription and depend on the adjuvant used jointly (as Resorcinol and nonionic surface active agent, for example polyoxyethylene oleyl ether and n-hexadecyl polyvinyl ether) with artificial increase intestines wall perviousness, and the enzyme inhibitors of being used jointly (as trypsin inhibitor, Isoflurophate (DFF) and the Trasylol of pancreas) is degraded with inhibitory enzyme.For oral, oral cavity contain in clothes, mucous membrane, the nose, stride film or designed being used to of rectal administration through lung, vagina and comprise that the prescription that the combination of albumen and antibody hydrophilic reagent and at least two kinds of tensio-active agents is sent instructs in U.S.6, in 309,663.The active constituent compound that is used for oral solid dosage can be mixed with at least a additive, and described additive comprises sucrose, lactose, Mierocrystalline cellulose, mannitol, trehalose, raffinose, maltose alcohol, dextran, starch, agar, arginates, chitin, chitosan, pectin, tragacanth gum, Sudan Gum-arabic, gelatin, collagen, casein, albumin, synthetic or semi synthetic polymer and glyceryl ester.These formulations also can contain the additive of other types, as inert diluent, such as the lubricant of Magnesium Stearate, p-Hydroxybenzoate, such as the sanitas of Sorbic Acid, xitix, alpha-tocopherol, such as the antioxidant of halfcystine, disintegrating agent, tackiness agent, thickening material, buffer reagent, sweeting agent, perfume compound, perfuming agent etc.
Tablet and pill can further be processed as the enteric coating preparation.Being used for oral liquid preparation comprises emulsion, syrup, elixir, suspension and allows medical pharmaceutical solutions.These preparations can contain the inert diluent that is generally used for described field, as water.The liposome as drug delivery system (the U.S. patent No. 4,239,754) that is used for Regular Insulin and heparin has also been described.Recently, used the artificial polymer microballoon of kilnitamin (proteinoid) to come delivering drugs (the U.S. patent No. 4,925,673) already.In addition, be described in the carrier compound in the U.S. patent No. 5,879,681 and the U.S. patent No. 5,5,871,753 and be used for the oral delivery biologically active agent and be all known in the art.
Mucous membrane prescription and administration
The prescription that is used for oral biologically active agent is encapsulated in one or more biocompatible polymkeric substance or multipolymer vehicle, preferably, be encapsulated in biodegradable polymer or the multipolymer, microcapsule is provided, because the suitable size of the microcapsule that produced, make this medicament arrive and be the folliculus lymph aggregate of animal, perhaps be called " peyer's patches " or " GALT " absorbs, and because medicament has passed through gi tract, so not loss of curative effect.Similarly folliculus lymph aggregate is found in segmental bronchus (BALT) and the large intestine.Above-mentioned tissue is commonly referred to as the relevant lymphoreticular tissue (MALT) of mucous membrane.In order to absorb by mucomembranous surface, composition and the method for using at least a anti-IL-6 antibodies comprise the emulsion that contains a large amount of submicron particles, mucoadhesive macromole, bioactive peptide and continuous water, promote absorption (the U.S. patent No. 5 by the mucous membrane adhesion that realizes emulsion grain by mucomembranous surface, 514,670).Be suitable for that the mucomembranous surface that emulsion of the present invention uses can comprise cornea, conjunctiva, contains clothes, hypogloeeis, nose, vagina, lung, stomach, intestines and rectal administration approach.The prescription that is used for vagina or rectal administration such as suppository can contain vehicle, for example polyalkenyl ethylene glycol, Vaseline, cocoa beans wet goods.The prescription that is used for intranasal administration can be solid-state and contain vehicle such as lactose, maybe can be the aqueous solution or the oily solution of nasal drop.For containing the clothes administration, vehicle comprises (the U.S. patent No.s 5,849,695) such as carbohydrate, calcium stearate, Magnesium Stearate, pre-gelledization (pregelinatined) starch.
Through skin prescription and administration
For percutaneous dosing, at least a anti-IL-6 antibodies is encapsulated in the delivery apparatus, for example liposome or polymer nano granules, particulate, microcapsule or framboid (unless otherwise mentioned, being called particulate jointly).Known multiple suitable device, comprise by synthetic polymer, polyhydroxy-acid for example, as poly(lactic acid), polyglycolic acid and their multipolymer, polyorthoesters, polyanhydride and polyphosphonitrile, and natural composition, the particulate (the U.S. patent No. 5,814,599) made of collagen, polyamino acid, albumin and other albumen, alginate and other polysaccharide and their combination for example.
Long term administration and prescription
Be desirably in and send compound of the present invention in prolonged for some time to the experimenter, for example, single-dose continues a week to year.Can utilize multiple slowly-releasing, long-acting or implantation formulation.For example, formulation can contain the pharmaceutically acceptable nontoxic salt that has the compound of low solubility in body fluid, for example, (a) has a acid salt such as the polyprotonic acid of phosphoric acid, sulfuric acid, citric acid, tartrate, tannic acid, pamoic acid, Lalgine, polyglutamic acid, naphthalene list or disulfonic acid, polygalacturonic acid etc.; (b) have multivalent metal cation, or have as N the formed organic cations salt of N '-dibenzyl-quadrol or quadrol such as zinc, calcium, bismuth, barium, magnesium, aluminium, copper, cobalt, nickel, cadmium etc.; Or (c) (a) and combination (b), as the tannic acid zinc salt.In addition, compound of the present invention or preferably undissolved relatively salt, those salt of describing just now for example, can with as the sesame oil that is suitable for injecting be formulated as for example Stearinsaeure alumina gel of gel.Particularly preferred salt is zinc salt, tannic acid zinc salt, embonate etc.The slow release long-acting prescription that is used for injecting of another kind of type should contain and be disperseed to be used to be encapsulated in compound or salt slow degraded, avirulent, nonantigenic polymkeric substance, described polymkeric substance is the polylactic acid/polyglycolic acid polymkeric substance for example, as the U.S. patent No. 3,773, described in 919.Compound or preferably undissolved relatively salt, for example above-mentioned those salts also can be formulated as the agent of cholesterol matrix silicone rubber plate, particularly use for animal.Extra slowly-releasing, long-acting or implantation prescription, be known in (the U.S. patent No. 5 in the document as gas or liquid fatty body, 770,222 and ＂ Sustainedand Controlled Release Drug Delivery Systems ＂, J.R.Robinson compiles., Marcel Dekker, Inc., N.Y., 1978).
The present invention has obtained general the description, will be easier to understand the present invention with reference to following examples, and described embodiment provides explanation as an example but not is intended to restriction.
Indication
Rheumatoid arthritis (RA)
Rheumatoid arthritis (RA) is a kind of chronic SIDA with autoimmunization feature.Can explain many features of RA by the IL-6 effect of imbalance.
May relevant IL-6 effect comprise the effect as B cell differential factor among the RA, induce the polyclone hypergammaglobulinemia and produce autoantibody (Rheumatoid factors, polyclonal) by IL-6; Promote cytotoxic T cell to grow, consistent with IL-2; Produce acute phase protein (CRP, SAA, Fibrinogen) by the liver cell stimulating activity; The osteoclast activation causes osteoporosis and bone destruction around the joint; By the effect as the megalokaryocyte differentiation factor, induced platelet increases; And regulate VEGF, and therefore may take place by blood vessel, consistent with IL-1 β and TNF α.
In RA, stimulate RA synovia inoblast to produce IL-6 by TNF α or IL-1, and in synovial membrane liquid (SF) and serum, exist with high density.Between the clinical/lab index of serum level and disease activity, exist dependency.
In some clinical studyes of activity RA patient, studied anti-IL-6/IL-6R mAbs.Result up to now is summarized as follows.
The I/II phase is studied
In these researchs at the beginning, use mouse-anti IL-6mAb (BE-8) continuous 10 day every day in 5 RA patients, and improve relevant (9) (Wendling, D with of short duration clinical and laboratory; Deng .1993 J.Rheumatol.20:259).The test anti-IL-6R of humanization (80kDa) mAb (MRA in studying in some RA patients' the I/II phase; Chugai).In these researchs at the beginning, by on every Mondays or twice intravenous infusion 1-50mg dosage use MRA, the continued treatment in 50mg/ week continues nearly 6 months.In the acute phase measurement, proteins C reactive (CRP) and Fibrinogen, low-heat, fatigue and clinical score be morning stiffness and swelling and the sharply minimizing of tenderness joint counting for example.Be also noted that anaemia, thrombocythemia and hypergammaglobulinemia improve.After research in, the MRA by single intravenous infusion 0.1-10mg/kg dosage handles 45 patients, has produced the trend that clinical score is improved on higher dosage level, adds the minimizing of acute phase reactant.
The II phase is studied
In these researchs at the beginning, handle 15 patients (dosage level 2,4 or 8mg/kg, weekly twice, continued for 24 weeks), and show that in 13/15 week in 24 weeks ACR20 replys, have the CRP/ serum amyloid A protein (SAA) of normalizing with MRA.Although mainly do not pay close attention to acute security, nearly 2/3 patient shows the LDL cholesterol levels of obvious increase.In studying in the ensuing II phase, (per 4 weeks give dosage level 4 or 8mg/kg to handle drug-fast 164 the RA patients of DMARDs by intravenous infusion with placebo or MRA, continue 3 months), and respectively for placebo, 4 and 8mg/kg show 11%, 57% and 78% ACR20 response rate in the 12nd week.At last, 359 activity RA patients accept independent MTX (10-25mg/ week), independent MRA (dosage level 2,4 or 8mg/kg were administered once by intravenous infusion in every month) or MTX+MRA.Reply mensuration by ACR20, compare with independent MTX, MRA and MRA+MTX are more effective.
Systemic lupus erythematosus (SLE)
SLE is a kind of chronic, fatal, autoimmune disease of possibility with multiple protean performance.Nosetiology the unknown.A sign of this disease relates to the autoantibody of the anti-multiple self antigen of B cell hyperproliferation, activation and generation.
IL-6 induces the B cytodifferentiation to be the antibody forming cell.In SLE, increased the generation of autoantibody (ANA, anti-dsDNA) by these antibody forming cells and immune complex deposit.IL-6 has promoted the cytotoxic T cell growth, has increased the liver acute phase reactant, mesangial cell propagation, keratinocyte growth, megalokaryocyte differentiation and thrombosis.
In SLE patient and mouse SLE model, the IL-6 level all raises.Proved that the whole end of the IL-6 receptor-inducible B cell that is combined on the B cell is divided into autoantibody and produces cell.Linker-Israeli etc. (Linker-Israeli M etc., 1991 J Immunol 147:117-123) have proved when using the neutralizing antibody of anti-IL-6, have reduced the generation of spontaneous polyclonal antibody.People such as Kitani (Kitani A, Deng, 1992 Clin Exp Immunol 88:75-83) doubled that the T cell produces and compared the SLEB cell with contrast B cell to have five times of more IL-6 and produce and supported these discoveries by proof external IL-6 in the SLE culture.Yet the pathology of autoantibody produce the effect that is not subject to IL-6 separately in SLE.Also studied the effect of IL-6 acceptor.People such as Nagafuchi prove with respect to normal B cell, IL-6 acceptor adjusted in most of SLEB cells.Anti-IL-6 receptor antibody has suppressed the whole end of these B cells and has been divided into the antibody forming cell.Also determined the effect of solubility IL-6 acceptor among the SLE.
Type ii diabetes (T2DM)
Insulin resistance (impaired insulin action) and impaired beta cell function (pancreas beta cell excreting insulin functional defect) are considered to the major cause of T2DM development.Insulin resistance shows as surrounding tissue and can't fully respond Regular Insulin and excite, and causes glucose level to increase thus.After insulin resistance and glucose level increase, at the Regular Insulin of the early stage pancreas beta cell of disease compensatory supersecretion.When T2DM made progress, the ability of beta cell excreting insulin had reduced.
Cause the inherent mechanism of insulin resistance not understand as yet.It is obesity that a kind of and T2DM develops common maximally related illness, and losing weight of appropriateness can both have been improved T2DM patient's glucose level significantly even.
Obesity and insulin resistance/hyperinsulinemia reduce with dyslipidemia, glucose tolerance and the hypertension common is characterised in that to have the illness that is called metabolism syndrome.Metabolism syndrome makes individuality easily develop into capillary blood vessel and great vessels change to the organic growth of T2DM, can cause cardiovascular (CV) disease and final dead.Thought already that obesity, insulin resistance and hyperinsulinemia most possibly got in touch T2DM and CV disease.
Fatty tissue has been accredited as the metabolic major organs of a kind of adjusting, is the energy storage storehouse and the endocrine organ of the many molecules relevant with the insulin sensitivity adjusting of secretion.Except that Leptin, phylaxin, adiponectin and TNF α, fatty tissue secretion IL-6 thinks to have contact between obesity, inflammation, T2DM and cardiovascular (CV) disease.
Proved between obesity and the IL-6 level and had positive correlation.The fatty tissue that increases in might obesity can provide continuing to increase of round-robin IL-6, by promoting the insulin sensitivity tissue inflammation can reduce insulin sensitivity or by disturbing activity and the protein expression relevant with the insulin signaling cascade to cause insulin resistance.Data all have existence in the external and body of support or opposition IL-6 latent effect in the insulin resistance development.
Utilized the vitro data of the well-defined cell system that comprises liver (HepG2) (44), fat (3T3L1) or isolating Islet cells to show that IL-6 exists direct negative effect respectively to insulin signaling conduction, glucose uptake and insulin secretion.On the other hand, the data that skeletal muscle examination of living tissue experiment is obtained think that IL-6 can increase glucose uptake in employed muscle.
For data in the body that concerns between IL-6 level and the insulin sensitivity are same mixing.Perhaps, IL-6 crosses expression or removes the active inhibition fully of model proposition IL-6 does not fully have useful effect.For example, in the non-obese diabetes of transgenosis (NOD) mouse, people IL-6 crosses to express and has delayed the outbreak of diabetes and prolonged existence.In addition, think that from the data of no IL-6 mouse IL-6 may work in energy balance is regulated, because these animals are developed into the glucose level of obesity and Geng Gao slightly late.
In the people, naturally occurring sudden change causes the increase of IL-6 secreting rate in the IL-6 promoter region.This sudden change is all relevant with minimizing with the increase of insulin sensitivity.
In another group experiment, assessed the effect of external source IL-6 already.In normal experimenter, IL-6 uses and causes the glucose level increase and do not influence plasma insulin concentration, otherwise, in the cancer patients, add IL-6 and increased glucose clearance.In addition, studied dependency between IL-6 level and the insulin resistance already.Think masculinity and femininity that from the data that these experiments obtain higher IL-6 cyclical level is relevant with higher insulin resistance, although cause-effect relationship still need be determined.
Shown already that IL-6 played an important role in the relevant insulin resistance development of obesity.Yet, exist and support or oppose data in the inconsistent external and body of its latent effect in insulin resistance.
Osteoarthritis (OA)
OA is a kind of chronic, osteoarthritis disease, is characterised in that loss joint cartilage and relevant subchondral bone change.Although observed inflammation in various degree in arthroscopy or in the synovial biopsy sample, this disease mainly is not an inflammation.On the contrary, it is regarded as originating from chondrocyte and/or scleroblast metabolism change.TNF, IL-1 and IL-6 change maximally related cytokine with these.
Although level is lower than inflammatory arthropathy in fact, in OA patient's synovia, can detect IL-6 (Bertazzolo .1994 Agents and Actions 41:90-92 such as N.).IL-6 is considered to the main stimulator of a kind of liver acute phase protein synthetic, even after the relation of considering between known CRP and the obesity, there be still relevant (Mohtai .1996 J Orthopedic Research 14:67-73 such as M.) in the CRP level with knee OA.
IL-6 expresses in the chondrocyte of OA cartilage, but does not express (Sowers .2002 Osteoarthritis and Cartilage 10:595-601 such as M.) in the chondrocyte of normal cartilage.In of the experiment of test mechanical stress to the effect of external chondrocyte's cytokine-expressing, the flow-induced shearing stress significantly adjusted IL-6mRNA and albumen.It may be caused by mechanical load that IL-6 in this hint OA cartilage expresses.Respond to the effect of IL-1, also can produce IL-6 (Dozin .2002 Matrix Biology 21:449-459 such as B.) the chondrocyte.
In other experiments, IL-6 and sIL-6R cause person joint chondrocyte's protein-polysaccharide synthetic of isolated culture to suppress jointly, although compare this effect not quite (Guerne .1999 Matrix Biology 18:253-260 such as P.) with IL-1.
Chronic obstructive pulmonary disease (COPD)
COPD is a kind of morbid state that flow limitation can't reverse feature fully that has.Flow limitation is normally progressive, and with lung to the reaction of the abnormal inflammatory of deleterious particle and gas relevant .2001 Am.J.Respir Crit Care Med 163:1256-1276 such as () Pauwels RA.COPD is characterised in that acceleration normally fails with the pulmonary function of age growth.Delaying into, the type flow limitation has caused deformity and premature death.COPD be dead and disabled main diseases because of, but only just it is being carried out extensive studies .2003EurRespir J 22:672-688 such as () Barnes PJ. recently from cell and molecule angle.In COPD, chronic inflammatory diseases causes little airway constriction of fixed and alveolar to destroy (pulmonary emphysema).Inflammatory reaction is characterised in that pulmonary alveolar macrophage, neutrophilic leukocyte and the cytotoxic t-lymphocyte of accelerating and the release of multiple inflammatory mediator (chemokine, cytokine, somatomedin and lipid).High-caliber oxidative stress can enlarge inflammation.Also increased elastolysis, and evidence show and relate to some Proteinase, bone marrow serines.Inflammation among the COPD and proteolysis are the expansion to the normal inflammatory reaction of cigarette smoke.As if different with asthma is, inflammation has resistance .2003 such as () Barnes PJ. to corticosteroid.
In animal model, bacterial endotoxin or lipopolysaccharides (LPS) and cigarette smoke expose the IL-6 that has induced neutrophilia and increased and produce .2000 AJPLCMP 279:L895-902 such as () Underwood D.C. in bronchoalveolar lavage (BAL) liquid.IL-6 has crossed induced expression pulmonary emphysema .2000 AJRCMB 22:289-295 such as () Kuhn HI Ch. in mouse lung.In the people, the polymorphonuclear cell counting retrocorrelation among the forced volume,expiratory (FEV1) in a second and IL-6, IL-8 level and BAL .1999 Eur Respir J 14:1015-1022 such as () Soler N..Level of tnfalpha in plasma, IL-6 and CRP level increase .1998 Respir Med 92:993-999 such as () Yasuda N. in middle severe COPD experimenter.
The acute exacerbation of COPD is defined as the lasting deterioration of patient's illness, from steady state to changing day by day away from normally, i.e. acute attack and need routine medication change (.2003 Eur Respir J 21:Suppl.41 such as Burge S., 46s-53s).Although the symptom that increases and the pulmonary function of deterioration are to be in hospital (in the U.S. about annual 500,000 people) common cause, but intrinsic cell and molecular mechanism still do not obtain broad research as yet, and be short in understanding (Wedzicha, J.A.2002 Chest 121:136S-141S).Acute exacerbation is for a long time sustainable and quality of life produced deep effect, also can quicken progress .2003 PuIm Med9:117-124 such as () Soto FJ. of COPD.Respiratory tract infection is that COPD worsens the modal cause of disease.These infection of great majority are caused by bacterium, but wherein many are by due to the virus infection, particularly rhinovirus infection .2003 such as () Soto FJ..Environmental factors, atmospheric polluting material and temperature also can work.
During worsening, neutrophilic leukocyte in COPD patient's sputum and IL-6, IL-8, TNF α and LTB4 concentration increase.Severe COPD patient is easy to frequent deterioration (annual 3 times or more times deterioration) in some.Even when COPD stablizes, this group patient (" frequently deterioration person ") still has the IL-6 of higher level and the SLPI of lower level (.2000 Thorax 55:114-120 such as Bhowmik A.; .2001Thorax 56:36-41 such as Gompertz S.; .2001 Eur Respir J 17:1112-1119 such as Gompertz S.).
Many other mechanism, for example oxidative stress and clusters of bacteria are equally also relevant with the physiopathology that COPD worsens.
Embodiment 1: clone and expression IL-6 antibody in mammalian cell
Typical mammalian expression vector contains promoter element, antibody coding sequence and Transcription Termination and the required signal of transcript polyadenylation of at least one regulating mRNA transcription initiation.Extra element comprises enhanser, Kozak sequence and supplies the donor of RNA montage and the intervening sequence of acceptor site flank.Use is from the early stage and late promoter of SV40, comes freely the retroviral length terminal repetition (LTRS) of RSV, HTLVI, HIVI and the early promoter of cytomegalovirus (CMV) can realize efficiently transcribing.Yet, also can use cell element (as the human actin promotor).Be applicable to that putting into practice expression vector of the present invention for example comprises, carrier such as pIRES1neo, pRetro-Off, pRetro-On, PLXSN or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).Mammals and other host cells of being fit to comprise people HeIa, 293, H9 and Jurkat cell, mouse NIH 3T3 and C127 cell, Cos 1, Cos 7 and CV 1, quail QC1-3 cell, mouse Lcell and Chinese hamster ovary (CHO) cell.Perhaps, gene can be expressed in containing the stable cell lines that is integrated into the gene in the karyomit(e).The use selective marker for example cotransfection of dhfr, gpt, Xin Meisu or Totomycin makes the evaluation of transfectional cell be able to possibility with separating.
Also can increase rotaring redyeing gene to express a large amount of encoding antibodies.DHFR (Tetrahydrofolate dehydrogenase) mark is used to develop the clone of the goal gene that carries hundreds of even several thousand copies.Another kind of useful selective marker is that (Murphy is etc., Biochem.J.227:277-279 (1991) for glutamine synthase (GS); Bebbington, etc., Bio/Technology10:169-175 (1992)).Utilize these marks, allow mammalian cell growth and selecting in selecting substratum have the cell of high resistance.These clones contain and are integrated into chromosomal amplification gene.Chinese hamster ovary (CHO) and NSO cell are through being usually used in producing antibody.
The strong promoter (LTR) (Cullen, etc., MoI.Cell.Biol.5:438-447 (1985)) that expression vector pC1 and pC4 contain Rous sarcoma virus adds CMV-enhanser fragment (Boshart, etc., Cell 41:521-530 (1985)).Be beneficial to the clone of goal gene such as the multiple clone site that has restriction enzyme cleavage site BamHI, Xbal and Asp718.Except that 3 ' intron, carrier contains the polyadenylation and the termination signal of rat proinsulin protogene.
Clone and expression in Chinese hamster ovary celI
Carrier pC4 can be used for expressing IL-6 antibody.Plasmid pC4 is the derivative of a kind of plasmid pSV2-dhfr (ATCC preserving number 37146).This plasmid contains the mouse DHFR gene that is subjected to the control of SV40 early promoter.Can be by being supplemented with the selection substratum of chemotherapeutic methotrexate (as alpha minus MEM, Life Technologies, Gaithersburg, MD) middle culturing cell is selected the active Chinese hamster ovary of shortage dihydrofolic acid or other cells with these plasmid transfections.Existing numerous documents put down in writing amplification DHFR gene in anti-methotrexate (MTX) cell (referring to as F.W.Alt, etc., J.Biol.Chem.253:1357-1370 (1978); J.L.Hamlin and C.Ma, Biochem.et Biophys.Acta 1097:107-143 (1990); And M.J.Page and M.A.Sydenham, Biotechnology 9:64-68 (1991)).Because the amplification of DHFR gene, thereby the cell of being grown under the MTX concentration that increases demonstrates resistance to this medicine by excessive generation target enzyme-DHFR.If second gene is connected in the DHFR gene, then coamplification is also crossed expression usually.This method known in the art can be used for developing the clone that carries more than the amplification gene of 1,000 copy.Afterwards, when discarding methotrexate, obtain to contain the clone of the amplification gene in the one or more karyomit(e)s that are integrated into host cell.
Except that long terminal repetition (LTR) strong promoter of Rous sarcoma virus, such as people's beta-actin promotor, SV40 is early stage or late promoter or come from other retroviruss such as efficient promoter that the length of HTV and HTLVI is terminal repetition also can be used for expressing.The Tet-Off of Clontech and Tet-On gene expression system and similar system can be used for IL-6 antibody expression (M.Gossen and H.Bujard, Proc.Natl.Acad.Sci.USA 89:5547-5551 (1992)) with regulative mode in mammalian cell.For the polyadenylation of mRNA, can use other signals equally, as signal from human growth hormone or globin gene.Also can with such as the basis of the selective marker cotransfection of gpt, G418 or Totomycin on select to carry the stable cell lines that is integrated into the goal gene in the karyomit(e).When being to begin, its favourable part uses more than a kind of selective marker, as the G418+ methotrexate.With digestion with restriction enzyme plasmid pC4, then by operation known in the art Roll Phosphoric acid esterase dephosphorylate.Then from 1% sepharose, isolate carrier.
According to known method steps, use the dna sequence dna of the complete IL-6 antibody of coding, as respectively corresponding to the HC of IL-6 antibody of the present invention and the sequence shown in SEQ ID NOS:98 and 96 of LC variable region.In this construct, also used the isolating nucleic acid of the suitable human constant region (being HC and LC district) of coding.
The carrier that connects isolating variable region and constant region coding DNA and dephosphorylate then with the T4 dna ligase.Transformed into escherichia coli HB 101 or XL-1 large cortical cells and use restriction endonuclease analysis for example to identify to contain the segmental bacterium that inserts among the plasmid pC4 then.
Chinese hamster ovary (CHO) cell that will lack active DHFR gene is used for transfection.Utilize lipofection reagent, with the expression plasmid pC4 of the plasmid pSV2-neo cotransfection 5 μ g of 0.5 μ g.Plasmid pSV2neo contains dominant selectable marker-the come from neo gene of Tn5, and coding is given the enzyme to the one group of antibiotics resistance that comprises G418.With cell inoculation in the alpha minus MEM that is supplemented with 1 μ g/ml G418.After 2 days, make the cell trypsinize and be seeded in hybridoma clone dull and stereotyped (Greiner, in Germany) be supplemented with 10,25 or the alpha minus MEM of 50ng/ml methotrexate+1 μ g/ml G418 in.After about 10-14 days, make the mono-clonal trypsinize, be seeded in then in 6-hole Petri dish or the 10ml flask, used the methotrexate (50nM, 100nM, 200nM, 400nM, 800nM) of different concns.Then the clone that will under the methotrexate of maximum concentration, grow transfer to contain in addition the new 6-hole flat board of the methotrexate (1mM, 2mM, 5mM, 10mM, 20mM) of greater concn in.Repeat identical operations up to obtaining till the clone who grows under the 100-200mM concentration.For example analyze the expression of the gene product of expectation by SDS-PAGE and western blotting or reversed-phase HPLC analysis.
Embodiment 2-makes up and the screening anti-IL-6 antibodies
Make up and screen activated IL-6 antibody variants (clone AME-A9).In the elsewhere of this embodiment and this paper, except that CDRH1 was Kabat and Chothia definition sum, CDRs was the Kabat definition.The length of CDRH2 is to make up two independent libraries to cover the necessary length in whole zone.Further identify to the purpose cloning and sequencing and by ELISA with based on the assay method of cell, and measure kinetic constant.
The example that the IgGs of use purifying carries out ELISA is shown among Fig. 9.ELISA uses Costar 3366 microtiter plates that are coated with the anti-people κ of goat antibody usually.Under 22 ℃ in the Fab (or IgG) of incubation dilution in the hole of bag quilt 1 hour.Then with PBS, 0.1%Tween 20 washing holes and add the biotinylated IL-6 incubation 1 hour of 200ng/ml.After the washing, add the alkaline phosphatase conjugate of NeutrAvidin, and 22 ℃ of following incubations 1 hour.Add colorimetric substrates and measure bonded IL-6 in a large amount of washings back.The modification of a kind of ELISA has comprised that behind biotinylated IL-6 incubation the washing steps that prolong are as the washing step of 18 hours prolongations under 37 ℃ in PBS, 0.01%BSA beaker.
The reactive IgG monoclonal antibody of people's engineering IL-6 that some the present invention produced has 1 x 10 9-9 x 10 12Affinity costant.The high-affinity of these people's engineering monoclonal antibodies makes them can be applicable to disease, pathology or the application of relevant treatment of conditions of IL-6-dependence.
Obtain multiple different people's through engineering approaches anti-IL-6 antibodies variant by the one or more CDR district that changes antibody.Following table 3 has generally been shown the useful sudden change of being found (amino acid change is with respect to the AME-A9 variant) in individual CDR library.In addition, following table 13 has shown the light chain in the replacement site (being labeled as " X ") with possibility and the aminoacid sequence of heavy chain CDRs.
Make up " combination " library based on the best clone (being variant) who in individual CDR library, is found.Table 4 has been listed the sudden change that is included in " combination " library.Screen and identify combinatorial library as mentioned above.Six preferably among the clone sudden changes found be shown among the following table 5A, the serial ID of the CDRs among these clones number is shown among the table 5B simultaneously.
In based on the assay method of cell, measure anti-IL-6 IgGs
Test inosculating antibody IL-6 and the anti-IL-6 of people's through engineering approaches (clone AME-19a) antibody stops the ability of IL-6 dependent cells system growth.The 7TD1 cell is layered in the Costar3610 96 hole flat boards with 200 cells/well.The antibody that dilutes in the IMDM substratum is added in the hole, add people IL-6 to 500pg/ml final concentration subsequently and flat board in the tissue culture thermostat container incubation 64-72 hour.At that time, add 50 μ l cell lysis buffer solution from ATPlite test kit (Packard Bioscience) in all holes, and dull and stereotyped 3 minutes of concussion.Add 50 μ lATPlite substrates and shake the flat board 1 minute of covering.On photometer, measure chemoluminescence.
Based on the results are shown among Figure 10 of raji cell assay Raji, has the calculating EC shown in the following table 6 50Value.The EC of chimeric anti-IL-6 antibodies 50Value is 2.7 x 10 -11M (4.09ng/ml), the EC of the anti-IL-6 of people's through engineering approaches (clone AME-19a) antibody 50Value is 2.7 x 10 -12M (0.41ng/ml).The EC of people's engineered antibody 50Value shows about 10 times of improvement, although at EC 50In the value, comprise intervening value, might obtain about 10 times-Yue 60 times improvement.
The binding kinetics of the anti-people IL-6 of embodiment 3-people engineering antibody
Elisa assay has confirmed that purifying relies on mode in conjunction with IL-6 from the antibody of these host cells with concentration.In this case, measure the avidity of antibody to its related antigen (epi-position).Utilize BIAcore analysis and KinExA 3000 instruments to obtain quantitative binding constant.The result shows that the avidity of some engineering monoclonal antibodies is very high, has 1 x 10 -9-3 x 10 -14K D
Used anti-people IL-6 monoclonal antibody (AME-A9, AME-A16, AME-18a, AME-20b, AME-22a and AME-23a) and detect combining of IL-6 and solubility IL-6 acceptor-sIL-6R as the enzyme immunoassay (EIA) of the CNTO 328 of positive control.Solubility human il-6 receptor-sIL-6R and recombinant human IL-6 obtain from R﹠amp; D Systems (Minneapolis, MN) (cat. no is respectively 227-SR-025 and 206-IL-010).(the H+L chain) that the anti-human IgG of goat-horseradish peroxidase connects obtains from JacksonImmunoresearch (West Grove, PA) (cat. no 109-035-003).Hydrogen peroxide and OPD sheet obtain from Sigma (St.Louis, MO) (cat. no is respectively H-1009 and P-8287).
The enzyme-linked immunoassay that the anti-IL-6mAb mixture of sgp80/TL-6/ forms
(MA) (goods catalogue numbering 9018) spends the night for Corning/Costar, Acton by the CostarEIA flat board with sIL-6R (10 μ g/ml are dissolved among the PBS, 100 μ l/ holes) bag down at 4 ℃.With the 0.15M salt water washing flat board that contains 0.02% (v/v) Tween 20, and at room temperature with being dissolved in 1% among the PBS (w/v) BSA (200 μ l/ hole) closed pores 1 hour.Washing hole once more, then at room temperature in turn with 200ng/ml people IL-6 (the 100 μ l/ hole) incubation 1 hour that is dissolved among the PBS.Antibody is added in all holes with 10 times of serial dilutions by 10 μ g/ml initial concentrations, 100 μ l/ holes, at room temperature incubation is 1 hour.After the washing, at room temperature with the anti-human IgG of goat (H+L)-HRP-conjugate (10 μ g/ml in PBS) incubation hole 30 minutes.Washing hole, and add Citrate trianion-phosphoric acid salt substrate solution (0.1M citric acid and 0.2M sodium phosphate, 0.01% H in 100 μ l/ holes 2O 2With 1mg/ml OPD), at room temperature incubation is 15 minutes.(MolecularDevices Spectromax Plus, Sunnyvale CA) reads OD also to pass through the dull and stereotyped reading apparatus of automatic ELISA by the 4N sulfuric acid stopped reaction that adds 25 μ l/ holes 490
At room temperature with 200ng/ml IL-6 (100 μ l) and antibody (the 100 μ l) incubation 1 hour that originates in 10 times of serial dilutions of 10 μ g/ml, with the effect of test with the IL-6 of hIL-6 monoclonal antibody or CNTO328 preincubation.Then at room temperature with this preincubation mixture and sIL-6R incubation 1 hour, and at room temperature use the anti-human IgG of goat (H+L)-HRP-conjugate (10 μ g/ml are dissolved among the PBS) incubation 30 minutes, detect the anti-people IL-6 of sIL-6R/IL-6/ mixture.Other condition determinations are identical described in the previous paragraphs with it.
Research before shown when CNTO 328 by according to the sIL-6R seizure of the dull and stereotyped internal technology report of EIA institute bag quilt the time, it can detect IL-6.In addition, utilize EIA, AME-A9, AME-A16, AME-18a, AME-20b, AME-22a and AME-23a can detect IL-6 and rely on combining of mode and sgp80 (sIL-6R) with dosage.Resist-IL-6 antibody according to everyone engineering of CNTO 328 assessments.Yet sIL-6R and IL-6 bonded ability have been eliminated in any pre-incubation of IL-6 and these anti-hIL-6 monoclonal antibodies.
Measure the kinetic constant of anti-IL-6 IgGs
The KinExA 3000 Instrument measuring binding kineticses that use Sapidyne to make.In brief, people IL-6 covalent coupling on the alzactone pearl, and is detected combining of free IgG and pearl on instrument.To measure K D,, among the PBS, contain 0.5,1 or single test tube 3-4 days of the people IL-6 of the serial dilution of the IgG of 5pM constant density and minimizing at 20 ℃ of following incubations in 0.1% BSA.For each K DMensuration has been used 13 test tubes altogether.For example, use the chimeric anti-IL-6 antibodies of 5pM constant density, and with the IL-6 incubation of single test tube and 0-200pM.Carry out the incubation of other IgGs in a similar manner.Behind the incubation,, on KinExA 3000 instruments, measure the free IgG in each equilibrated sample according to manufacturer's operation instruction.As described in more detail below, use KinExA 3000 instruments to measure K by KinExA 3000 softwares DValue.
Single IgGs of kind of 200pM is mixed with the people IL-6 of 100-200pM to measure k On, by on KinExA 3000 instruments, measuring unconjugated IgG with the combining of people IL-6 of covalent coupling alzactone pearl.Along with the time is carried out a series of mensuration in the past.Use KinExA3000 software, resulting data are used to calculate k OnBy using formula K D=k Off/ k OnCalculate k OffThe kinetic constant of anti-IL-6IgGs generally is shown in Table 7.
Embodiment 4: external evaluation anti-IL-6 antibodies
Carry out in vitro study to identify sequence, epitope specificity, avidity and the biological activity of anti-IL-6 antibodies.
People's engineering mAb
Sequential analysis has confirmed that anti-IL-6 antibodies of the present invention (being embodied in different variant/clones) contains complete people's framework region.Table 5a shown with chimeric anti-IL-6 antibodies (being described in PCTWO 04/039826) and compared, and always has 10 amino-acid residues and change in the CDR1 of anti-IL-6 antibodies of the present invention (in different antibody variants), 2 and 3 heavy chain and light chain.
Epitope specificity
Similar epi-position on anti-IL-6 antibodies of the present invention and the inosculating antibody IL-6 antibody recognition people IL-6.These antibody not with obtain from R﹠amp; The commercial mouse anti human IL-6mAb competition of D Systems #MAB-206 hints that the epi-position of their identification is unique, is different from R﹠amp; The epi-position of the anti-IL-6mAb of D.Anti-IL-6 antibodies of the present invention and chimeric anti-IL-6 antibodies not with R﹠amp; D rat Anti-Human IL-6mAb competition.
By anti-IL-6mAb (the mouse anti human IL-6mAb of dull and stereotyped bonded, MAB-206, only be used as dull and stereotyped bonded mAb and catch people IL-6) (10 μ g/ml) catch people IL-6 (200ng/ml), and serial dilution anti-IL-6 antibodies of the present invention and chimeric anti-IL-6 antibodies, then as shown in the X-axle, adding on the flat board.Survey the OD that increases by along the Y-axle 490Mensuration combines with IL-6's.Anti-IL-6 antibodies of the present invention and chimeric anti-IL-6 antibodies all show dose rely on the combining of IL-6.
On the contrary, anti-IL-6 antibodies of the present invention and inosculating antibody IL-6 antibody competition be in conjunction with people IL-6, hints that these two molecules all have IL-6 and go up similar in conjunction with epi-position.Catch people IL-6 (200ng/ml) by dull and stereotyped bonded MAB-206 (10 μ g/ml).Add then as along the biotinylated chimeric anti-IL-6 antibodies of the anti-IL-6 antibodies of the present invention of the serial dilution shown in the X-axle and 50ng/ml to flat board.By streptavidin-chimeric anti-IL-6 antibodies of HRP detection of biological elementization and combining of IL-6, and be determined as along the OD of Y-axle 490Reading.
In addition, people's engineering shows the similar characteristic in conjunction with the sIL-6/sIL-6R mixture (Fig. 1) with chimeric antibody.Anti-IL-6 antibodies of the present invention is in conjunction with the sIL-6/SIL-6R mixture.Solubility IL-6 acceptor (sIL-6R) is coated on the flat board with 10 μ g/ml concentration.Add people IL-6 to flat board with 200ng/ml concentration then.Then add as along the anti-IL-6 antibodies of the present invention of the serial dilution shown in the X-axle or chimeric anti-IL-6 antibodies to flat board, and use the anti-human IgG of HRP-to detect and the combining of IL-6/sIL-6R mixture, and be determined as along the OD of Y-axle 490Reading.
In based on 7TD1 (IL-6 relies on mouse hybridoma system) cell proliferating determining, use generation to carry out the reactive test of cross species with the above-mentioned discovery of further confirmation from the condition supernatant liquor that contains IL-6 of the PBMCs of the different plant species of LPS and IFN γ-stimulation.In the 7TD1 cell proliferation that stimulates from the multiple primate species that comprises people, marmoset monkey, rhesus monkey, chimpanzee, macaque, baboon, stump-tailed macaque and thin,tough silk hair monkey (cotton topmonkey), people's engineered antibody of the present invention has shown neutrality condition supernatant liquor activity, compares with chimeric antibody also to have shown the reactive collection of illustrative plates (table 8) of similar cross species.
At last, when using the tryptic digestion method to carry out epitope mapping, observe for people's engineering and chimeric antibody, identical in conjunction with epi-position people IL-6 on, it is positioned at the amino-acid residue 168-184 (Fig. 3) of leap spiral D.Recently to have confirmed that residue 179 and 182 is that antibody of the present invention combines with IL-6 necessary for mutation analysis.In the presence of people's through engineering approaches anti-IL-6 antibodies, this epi-position (amino-acid residue 168-184) is differentiated to be positioned on the IL-6 surface that remains with deuterium.
Biological activity
Tire by the IL-6 neutralization of measuring people's through engineering approaches anti-IL-6 antibodies based on the bioassay method of 7TD1 cell.In the 7TD1 cell proliferating determining, compare people's through engineering approaches anti-IL-6 antibodies with chimeric anti-IL-6 antibodies and confirm that having 10 times of higher neutralizations tires.In the presence of people's through engineering approaches anti-IL-6 antibodies or chimeric anti-IL-6 antibodies or isotype contrast mAb of serial dilution, use the hIL-6 of 500pg/ml to stimulate the 7TD1 cell 72 hours.Cell proliferating determining is as the counting of the per second shown on the Y-axle.Error line is represented the SD of duplicate sample.The cell of the no IL-6 of filled circles representative; The open circles representative hIL-6 stimulated cells of 500pg/ml.
People's through engineering approaches anti-IL-6 antibodies also suppresses IL-6-inductive U937 cell MCP-1 (MCP-1) and produces (Fig. 3) and IL-6/IL-1 β-inductive HepG2 human liver cancer cell serum amyloid A protein (SAA) generation (Fig. 4).Fig. 3 has confirmed that people's through engineering approaches anti-IL-6 antibodies suppresses the U937 cell MCP-1 secretion that IL-6 stimulates.Handle 5 x 10 with the hIL-6 of 1ng/ml and people's through engineering approaches anti-IL-6 antibodies of serial dilution 5Individual cells/well 72 hours.By the existence of MCP-1 in the elisa assay cell culture supernatant liquid, analyze triplicate.
Fig. 4 has shown that people's through engineering approaches anti-IL-6 antibodies suppresses the HepG2 cell SAA secretion of IL-6 and IL-1 β stimulation.In the presence of people's through engineering approaches anti-IL-6 antibodies of serial dilution, stimulate 2.25 x 10 with the sIL-6R of hIL-6, the 200ng/ml of 100ng/ml and the IL-1 β of 1ng/ml 5Individual cell 24 hours.Pass through the existence of SAA in the elisa assay cell culture supernatant liquid then.
The Stat3 phosphorylation that IL-6 relies on
Carrying out immune precipitation determination blocks by the ability of IL-6 in conjunction with IL-6R and the caused signal cascade of gp130 with the evaluator through engineering approaches anti-IL-6 antibodies that is used for of testing the STAT3 phosphorylation that in the THP-1 cell of expressing gp130 on cell surface IL-6 is relied on.
To mAbs sterile filtration sterilization and under 4 ℃, be stored among the PBS.Use is from R﹠amp; DSystems (Minneapolis, recombinant human IL-6 MN) (206-IL-010) and sIL-6R (227-SR-025).RPMI substratum (11875-085), heat-inactivated foetal calf serum (16000-069), L-glutaminate (25030-081), non-essential amino acid (11140-050) and Sodium.alpha.-ketopropionate (11360-070) obtain from Invitrogen (Carlsbad, CA).Also used TBS (10mMTris, pH7.5,100mM NaCl).
THP-1 (a kind of people's acute monocytic leukemia clone that obtains from research cell bank (research cell banks)) is the mycoplasma feminine gender and does not contain bacterium after tested.In the RPMI substratum that contains 10% foetal calf serum, 2mM glutamine and 1mM Sodium.alpha.-ketopropionate, cultivate these cells.Cultured cell line or reach about 85% harvested cell when converging when culture.Cell just was split into 1:5 routinely in per 3 days.
For tyrosine phosphorylation, cell grows to 80-90% and converges in the T-225 flask.Discard substratum and use the fresh culture of serum-free to replace, be incubated overnight.Behind serum starvation, harvested cell from each flask, pelleting and with 20 x 10 of final concentration under every kind of condition 6Individual cell is resuspended in the 0.5ml serum free medium.
Substratum, anti-IL-6Ab (10 μ g/ml) and sIL-6R (0.2 μ g/ml) that RhIL-6 (0.1 μ g/ml) and following reagent: 0.5ml is independent were 37 ℃ of following preincubation 15 minutes.Add respectively then SIL-6R (0.2 μ g/ml) and anti-IL-6Ab (10 μ g/ml) to the cell of anti-IL-6Ab and sIL-6R preincubation in, 37 ℃ of following incubations 15 minutes.Then cell combines with substratum and IL-6/Ab/sIL-6R mixture as negative control, and 37 ℃ of following incubations 6 minutes.Washed cell twice in ice-cold TBS, and as 5.4 joint granular precipitations of described processing cell or be stored under-70 ℃.
For immunoprecipitation, containing whole protease inhibitor cocktail sheets (1697498, Roche, Basel, 1ml lysis buffer (50mM Tris Switzerland), pH7.5,300mM NaCl, 0.5% Triton-X-100) (T-9284, Sigma, St.Louis, MO) the middle granular precipitation of dissolved cell.Vortex cell 30 seconds and at-70 ℃ of following incubation 20-60 minutes.13,000rpm removed cell debris in centrifugal 20 minutes.By on the orbit determination mixing tank in 4 ℃ down with 2 μ g rabbit iggs (15006, Sigma, St.Louis, MO) add 50 μ l albumin A agarose (SC-2001, SantaCruz Biotechnology, Santa Cruz, CA) 1 hour precleaning sample of incubation is to reduce non-specific background dyeing.Removed the agarose pearl in centrifugal 5 minutes by 2500rpm.Transfer to clarifying lysate in the miniature centrifuge tube and on the orbit determination mixing tank in 4 ℃ down with anti-STAT3 (2 μ g/ml) (SC-7179, Santa Cruz Biotechnology) be incubated overnight, add 50 μ albumin A agarose pearls afterwards and on orbital shaker in 4 ℃ of following incubations 2 hours.Collected the agarose pearl in centrifugal 5 minutes by 2500rpm, and in ice-cold TBS, washing 5 times under 4 ℃.Then, with the agarose pearl be resuspended in the 40 μ l Laemmli sample buffers that add DTT (NP0007-465030, Invitrogen, Carlsbad, CA) in, and 95 ℃ of following heating 5 minutes.
Have running buffer (NP0002-465026, Invitrogen, Carlsbad, (Carlsbad CA) goes up to run down in 100V and resolved sample in 1 hour 3-8% NuPage Bis-Tris gel CA) for EA0375BOX, Invitrogen.Under 30V, use transfering buffering liquid (NP0006-465029, Invitrogen, Carlsbad, CA) run 1 hour with albumen transfer to nitrocellulose membrane (LC2001, Invitrogen, Carlsbad, CA) on.(California) middle closing membrane spends the night for Nestle, Glendale being dissolved in 10% skimmed milk powder of TBS-T under 4 ℃.At room temperature in TBS-T after the washing several times, on the orbital shaker under 4 ℃ with the anti-p-STAT3 Ab of mouse monoclonal of film and 1:1000 dilution in TBS-T (SC-8059, Santa Cruz Biotechnology, Santa Cruz, CA) incubation is 4 hours.Several times after the washing, then on the orbit determination mixing tank under room temperature with film and the anti-mouse IgG-HRP of donkey (1:1000) (SC-2318, Santa CruzBiotechnology, Santa Cruz, CA) incubation is 2 hours.Several times after the washing,, use ECLplus western blotting detection reagent and assay kit (RPN2108, Amersham Biosciences, Piscataway, NJ) test sample, and be able to developing by being exposed to the ECL film according to manufacturer's experimental program.Then, by film is immersed in 100mM DTT under 100 ℃, 2%SDS, 62.5% mM Tris-HCI stirred 30 minutes among the pH 6.7, peeled off Ab.The washing film also spends the night with the sealing of 10% skimmed milk powder in TBS-T then.Use anti-STAT3 (the 1:1000) (SC-7179 that is dissolved among the TBS-T down in 4 ℃, Santa Cruz Biotechnology) washing and incubation film are 2 hours, wash 5 times, afterwards with goat anti-rabbit igg-HRP (1:1000) (SC2030, Santa Cruz Biotechnology, Santa Cruz, CA) incubation is 1 hour, and uses ECLplus to detect.All films all have been stripped from Ab without any exception and have been existed to confirm that STAT3 is proteic with the STAT3 heavy label.
The result shows that people's through engineering approaches anti-IL-6 antibodies blocked the integral part (Fig. 5 A and 5B) of a kind of key in the stat3 phosphorylation-IL-6 signal path of IL-6-mediation.People's through engineering approaches anti-IL-6 antibodies (AME-19A) has suppressed IL-6/sIL-6R-inductive stat3 phosphorylation.In the THP-1 cell, detected recombinant human IL-6/sIL-6R-inductive stat3 phosphorylation (Fig. 5 B).The people's through engineering approaches anti-IL-6 antibodies (AME-19A) or the chimeric anti-IL-6 antibodies that add 10 μ g/ml have suppressed stat3 phosphorylation (Fig. 5 B) fully.Fig. 5 A shown corresponding to the different anti-IL-6 of people's through engineering approaches be cloned in exist in all samples analog quantity not by the stat3 albumen of phosphorylation.CNTO328 (or 328) refers to chimeric people-murine antibody (being also referred to as wild-type (WT)) as used herein, 150 refer to clone AME-22a, 143 refer to clone AME-23a, 140 refer to clone AME-20b, 136 refer to clone AME-19a, 130 refer to clone AME-18a, and 106 refer to clone AME-A16, and 104 refer to clone AME-A9.
Validity in the body of people's through engineering approaches anti-IL-6 antibodies
The validity of evaluator through engineering approaches anti-IL-6 antibodies in two different body inner models.At first, in measuring, the people IL-6-of mouse inductive matrigel blood vessel tests and compares the effect of people's engineering and chimeric anti-IL-6 antibodies.The people IL-6 of 200ng/ml is included in the matrigel bolt.Two matrigel embolisms are gone in every nude mouse.The winding of 6 mouse is subjected to 1,3 or the intravenous injection of 6mg/kg people's engineering or chimeric anti-IL-6 antibodies.Also PBS or isotype contrast mAb are applied to control group.Take out the matrigel bolt on the 7th day and pass through content of hemoglobin in the matrigel bolt, capillary blood vessel length and the generation of microvessel count flow measurement blood vessel.By measuring all three parameters, the result shows that people IL-6 (PBS group) has stimulated the blood vessel in the matrigel bolt model to take place.
People's through engineering approaches anti-IL-6 antibodies (AME-19A) has suppressed microvascular average quantity in the matrigel bolt.In addition, people's through engineering approaches anti-IL-6 antibodies (AME-19A) has suppressed microvascular mean length in the matrigel bolt.Equally, people's through engineering approaches anti-IL-6 antibodies (AME-19A) has suppressed the hemoglobin level in the matrigel bolt.
In addition, people's engineering (AME-19A) and chimeric anti-IL-6 antibodies all suppress the blood vessel generation that IL-6-mediates in the nude mouse in dose-dependently ground.Finally, under the highest proof load 6mg/kg, people's engineering and chimeric anti-IL-6 antibodies have shown comparable activity in suppressing the generation of IL-6-inductive blood vessel.Although as measuring by length of vessel and blood vessel quantity, chimeric anti-IL-6 antibodies has obviously suppressed the generation of people IL-6-inductive blood vessel under 3mg/kg, but under these dosage, do not detect significant difference on the statistics between people's engineering and the chimeric anti-IL-6 antibodies.
Developed a kind of extra body inner model and be used for of the effect of further evaluator through engineering approaches anti-IL-6 antibodies Balb/C mouse people IL-6-inductive acute phase reactant-serum amyloid A protein (SAA).Used in intravenous injection before the people IL-6 of 5 μ g/kg 4 hours, mouse is accepted people's through engineering approaches anti-IL-6 antibodies (Fig. 6) of 0.01,0.5 or 5mg/kg that abdominal injection uses.PBS and isotype contrast mAb are used as contrast.Inject the Serum SA A level of measuring in back 16 hours at IL-6.People's engineering and chimeric anti-IL-6 antibodies 0.5 and 5mg/kg under all suppressed in the Balb/C mouse people IL-6-inductive SAA significantly and produced, people's through engineering approaches anti-IL-6 antibodies has suppressed the SAA generation significantly under the lowest dose level of test.Yet, under all three kinds of proof loads, do not observe significant difference on the statistics (Fig. 6) between people's engineering and the chimeric anti-IL-6 antibodies.
Fig. 6 has shown that people's through engineering approaches anti-IL-6 antibodies suppresses people IL-6-inductive SAA and produces.The SAA mean value of every animal of each some representative, line is represented the mean value of all data points in every group.Carry out paired comparison, and use Tukey ' s 95% simultaneous confidence intervals, so as to control overall I type error ( *P＜0.001, *P＜0.05).
The treatment theoretical foundation of the anti-IL-6mAb of embodiment 5-
Rheumatoid arthritis. anti-IL-6mAb is to collagen-induced sacroiliitis (CIA)-one kind wind The effect of wet arthritis animal model
Disease model in the clinical precursor
In multiple body inner model already target IL-6.Use rat anti-mouse IL-6 antibody at the standard mouse model, or in primate model and people/mouse SCID model, use the anti-IL-6R of humanization (80kDa) mAb (MRA; Chugai).In the collagen-induced sacroiliitis of mouse (CIA), if early use (with after the collagen immunization the 0th or 3 day), then anti-IL-6 has effectively prevented disease, but after the time point use then invalid.People's synovial tissue is transplanted in people/mouse SCID transplantation model in the immunodeficiency type mouse therein, and MRA handles and causes tissue grafts to shrink and reduce inflammatory cell and osteoclast.In rhesus monkey CIA, MRA has suppressed the sacroiliitis development, and has improved the acute phase observed value.
In the CIA model, assessed the effect of the anti-mouse IL-6mAb of surrogate to disease progression.The result shows, as obvious reduction disease severity reflected, before disease is induced, use anti-mouse IL-6 and suppressed collagen-induced arthritic development significantly with 1mg/ mouse/all abdominal injections.With the 100 μ g II type bovine collagen intradermal injection afterbodys that are in the Freund's complete adjuvant (FCA), induce sacroiliitis in the DBA/1 LacJ mouse in 8 ages in week.Clinical monitoring mouse disease outbreak every day.Before CIA induced 2 days, after this anti-IL-6mAb or isotype contrast mAb that abdominal injection is used the 1mg/ mouse injected once weekly.Determine the sacroiliitis score based on swelling, erythema and joint deformity.
The histopathology data acknowledgement weekly the once anti-mouse IL-6mAb of abdominal injection improved the clinical observation of collagen-induced sacroiliitis parameter significantly.Compare with the animal that contrast mAb handles, all checked sacroiliitis parameters comprise that inflammatory reaction (synovitis and pannus form) and erosion change (corroding and whole articulation structure) and all improve significantly in the mouse that anti-mouse IL-6 handles.Anti-IL-6mAb has suppressed sacroiliitis on the histopathology level.Based on synovial membrane thickness synovitis is given a mark; Based on pannus degree pannus is formed marking with respect to the joint space; And based on the degree of invading cartilage and subchondral bone to corroding marking.
The loss of cartilage matrix protein obviously reduces in the mouse of handling with anti-mouse IL-6mAb.By the representative joint cut sections for microscopic examination cartilage matrix of Toluidine blue staining to the animal that obtains in research latter stage (the 53rd day) to handle from contrast and anti-IL-6mAb.
Little-CT analyzes the clinical observation of having supported that anti-mouse IL-6 treatment is worked on the level of progression of disease in individual joint.Compare with dominant slight soft tissue inflammation change in the anti-mouse IL-6 processing joint of animal, typical 3D CT image direct viewing has been shown occur in significant erosion change in the isotype contrast mAb treatment group.Use the representative animal of contrast mAb processing and the animal of anti-mouse IL-6mAb processing to experimentize.
Lupus. the effect of anti-IL-6 in NZB/W F1 mouse
Disease model in the clinical precursor
Mouse model exists SLE, and these are similar closely to the human disease.To the close similar B cell hyperproliferation to the human disease that studies confirm that of MRL/lpr and NZB/W F1 strain, autoantibody produces and immune complex deposit.Anti-IL-6mAb shows that can suppress autoantibody effectively produces, reduces proteinuria and improve animal survival in NZB/W F1 mouse.
In NZB/W F1 mouse, assessed the effect of the anti-mouse IL-6mAb of surrogate to the lupus morbidity.PRELIMINARY RESULTS has confirmed to use the generation (Fig. 7) that has suppressed anti-dsDNA autoantibody-a kind of main pathogenicity bo autoantibody in this disease model anti-22 weeks of mouse IL-6mAb with 1mg/ mouse/all abdominal injections.Compare with the animal that contrast Ab handles with salt solution, anti-dsDNA autoantibody level is lower consistently in the animal that anti-IL-6mAb handles in whole research.
As discussed above, Fig. 7 has shown that having suppressed the anti-dsDNA autoantibody by anti-IL-6mAb in NZB/W F1 mouse produces.The individual O.D. value that makes every kind of sample is to positive control serum normalization method and be expressed as the % positive control.The % positive control of every kind of sample of each some representative, line is represented the mean value of all data points in every group.Significant difference is expressed as *P＜0.01.
In addition, when the animal of a little subgroup was checked, anti-IL-6mAb had suppressed B-cell proliferation and has reduced renal impairment.Though the T cell proliferation between research different treatment group in latter stage does not have significant difference, but along with the past of time, particularly after 34 weeks, compare with the mouse of brine treatment, anti-IgM and anti-CD40 inductive B-cell proliferation reduce in the mouse that anti-IL-6mAb handles.This result is consistent with the anti-dsDNA autoantibody generation of the minimizing of above-mentioned report, has hinted for anti-IL-6mAb and has handled, and id reaction B cell may be direct main target spot.
Histopathological analysis shows that the animal capable in this research is divided into 3 class ephrosis severity groups (slightly, moderate and severe) (table 9).Pathology of renal disease in the NZB/W F1 mouse has shown Combination lymphoid hyperplasia and immune complex deposit in the glomerular basement membrane.
Develop into not serious ephrosis with the animal that anti-IL-6mAb handles.In the animal that anti-IL-6mAb handles, there are not Combination lymphoid hyperplasia and proteinosis around the blood vessel, but then develop into moderate and severe blood vessel Combination lymphoid hyperplasia and proteinosis on every side with the animal that salt solution and contrast Ab handle.In addition, compare with other two treatment group in the animal mesonephric glomerulus basilar membrane that anti-IL-6mAb handles and have slight immune complex deposit.Because the effect of these cells its key in SLE pathology, so carried out the mechanism analysis of further anti-IL-6mAb to B, T and macrophage function effect.
Type ii diabetes
Shown already that IL-6 played an important role in the insulin resistance development relevant with obesity.Yet its latent effect in insulin resistance had not only been supported but also opposed to data in the resulting up to now external and body.
Experiment in vitro
The body inner model that uses insulin sensitivity organizer's external model (for fatty tissue 3T3L1 cell, for liver cell HepG2 cell, for skeletal muscle C2C12 cell) and insulin resistance and T2DM is the db/db mouse for example, experimentized with understand better that IL-6 may have to insulin signaling conduction and Regular Insulin biological effect and for example glucose uptake, generegulation and relevant machine-processed effect of function.
Vitro data hint IL-6 in liver mainly conducts insulin signaling and works.The IL-6 of HepG2 cell handles the inhibition that causes insulin-induced Akt phosphorylation.This IL-6 is blocked by anti-IL-6 antibodies the restraining effect of insulin signaling conduction.Hinted that glucose metabolism change and insulin action are to drive insulin resistance and T2D reasons of development in the liver.Checked the effect of IL-6, to determine the mechanism of IL-6 among the T2D to insulin signaling conduction among 3T3 L1 cell (adipose cell lines) and the C2C12 (Skeletal Muscle Cell system).
3T3 L1. uses 3T3 L1 mouse adipose cell lines to experimentize.In the 3T3 L1 cell of 90% differentiation, assessed the effect of IL-6 to insulin-induced glucose uptake.In these experiments, suppressed insulin-induced glucose uptake consistently in 24 hours with the TNF α of 10ng/ml processing, the IL-6 of 20ng/ml is then without any effect.These data suggest IL-6 is not the main mechanism of the insulin resistance of IL-6 mediation to the activity of fatty tissue, and fatty tissue may be the main source that disturbs the IL-6 of insulin sensitivity in liver and the muscle afterwards.Use has obtained identical data from the primary human adipocyte of the differentiation in subcutaneous lipids storehouse.Use has been tested the effect of IL-6 to glucose uptake from the elementary adipocyte of interior fat storehouse people, because for the obesity relevant with insulin resistance, fat depot may be more relevant.
HepG2. select the HepG2 cell as the external representative of liver organization.The HepG2 cell is wherein to have shown before the Bel7402 of IL-6 to the effect of insulin signaling conduction already.In experiment, the IL-6 of 20ng/ml has blocked the crucial kinases in insulin-induced Akt phosphorylation-a kind of insulin signaling conduction path, observes after 60 minutes at incubation to have maximum effect; Consistent with institute's results reported in the scientific literature.
After 30,60,90 and 120 minutes, measure the Akt phosphorylation on the HepG2 cell that converges in the 10cm plate Central Asia at rh IL-6 (20ng/ml) incubation.During last 5 minutes of incubation, add 0.5nM, 1nM and 5nM Regular Insulin to induce the Akt phosphorylation.Use the RIPA lysis buffer dissolved cell of improvement, and utilize Ser-Phospho-Akt ELISA to measure the Akt phosphorylation.Utilize pAkt and Akt ELISA test kit (BioSource) to obtain the result.Handle at IL-6 the 60th minute, in the presence of the Regular Insulin (0.5-1nM) of physiological concentrations, to compare with control group, the Akt phosphorylation is suppressed～and 50%.Use the quantitative protein concentration of Pierce BCA protein determination kit.
The effect of IL-6 antibody
Measured people's through engineering approaches anti-IL-6 antibodies and suppressed the ability of IL-6 insulin-induced Akt-phosphorylation.People's through engineering approaches anti-IL-6 antibodies of 20 μ g/ml can suppress the IL-6 effect in the HepG2 cell.Fig. 8 A and 8B shown people's through engineering approaches anti-IL-6 antibodies exist or not in the presence of IL-6 to the effect of insulin-induced Akt phosphorylation.
Among the figure atop (Fig. 8 A), data are mean+/-SEM. ( *Significantly, compare P=0.029 with (+) Regular Insulin, IL-6; *Significantly, compare with (+) Regular Insulin+IL-6, P=0.02).IL-6 with 20ng/ml handled the inferior HepG2 cell that converges 60 minutes.During last 5 minutes that handle, add 1nM Regular Insulin and use the RIPA damping fluid dissolved cell of improveing.The elisa assay sample of Ser 473 phosphorylations by detecting Akt.All data are the total Akt normalization method to measuring by ELISA all.AME-19a handles and can recover normal Akt signal conduction.
(Fig. 8 B) shown representational western blotting among the figure down below.Band on the top comprise with IL-6 (20ng/ml 60 minutes, use 1nM Regular Insulin, 5 minutes), AME-19a (20ug/ml+/-IL-6 (20ng/ml, 60 minutes), use 1nM Regular Insulin, 5 minutes) or the sample of damping fluid processing.(pS473 Biosource) surveys trace with anti-phosphoric acid Ser/Akt antibody (upper area).Under band (peel off down above-mentioned trace and use anti-Akt heavily to survey) from BioSource shown and loaded equal protein in the per pass.
Method: in 100mm tissue culture ware, cultivate the HEPG2 cell until converging.Cell hunger is spent the night.Before IL-6 adds, about 30 minutes of pair cell incubation AME-19a (20ug/ml).Before adding cell to, incubation IL-6 (20ng/ml)+/-about 30 minutes of AME-19a (20ug/ml).In 37 ℃ of following pair cell incubation samples 60 minutes; Add 1nM Regular Insulin (final concentration) then to cell, at room temperature incubation is 5 minutes.Come washed cell 3 times with ice-cold PBS rinsing at once.Frozen plate is till being used for cracking.Use ELISA test kit (BioSource and Sigma) to measure phosphate Akt and total Akt.
Reference:JJ Senn, PJ Kover, IA Nowak and RA Mooney.Interleukin 6induces cellular insulin resistance in hepatocytes.Diabetes.51:3391-3399,2002.
Primary rat hepatocyte
Primary hepatocyte is that a kind of more relevant test I L-6 and anti-IL-6 antibodies (or other IL-6 antagonists) of being suitable for is to insulin signaling conduction and the Regular Insulin vitro system to hepatic glucose generation effect.Handle isolated cells in 5ng/ml IL-6 existence or not with Regular Insulin, and use ELISA to measure and western blot analysis is measured the phosphorylation of insulin receptor, IRS-1 (Figure 12 A) and Akt (Figure 12 B), measure that PI3 kinases (PDK) activates in the rat hepatocytes of Regular Insulin, IL-6 and/or IL-6mAb processing.In addition, check that IL-6 is to the associating effect of the IRS1/p85 of insulin stimulating (Figure 11 A and B).This experiment is carried out as follows:
In the flat board of 6 hole glue primordial coverings with primary rat hepatocyte (～2 months big) equilibrate overnight in the Hepatoczyme substratum.Second day, in DMEM-1% BSA-pennstrep, made cell hungry 6 hours; Then at 37 ℃ of following and hIL-6 (5ng/ml); Anti-IL-6 antibodies (AME-19a) (20ng/ml) or anti-IL-6 antibodies (AME-19a)+hIL-6 incubation 90 minutes.Before adding aforesaid combination, use anti-IL-6 antibodies (AME-19a) pretreatment cell 1 hour.Preincubation is also carried out in this combination before adding cell to.Add 5nM Regular Insulin (from BioSource) to cell, incubation 5 minutes; Extract cell then out and use BioSource extraction buffer+proteinase inhibitor cracking at once.Centrifugal lysate, 1:10 dilution supernatant liquor also is used for ELISAs (from Biosource) test.
IRS1/p85 associates:
The albumen (45 μ g) of equivalent is incubated overnight with the anti-IRS-1 polyclonal antibody of 2 μ g (from Upstate, product #06-248).Use albumin A pearl immunoprecipitation sample 1 hour then, and be used for SDS-PAGE with 3x sample buffer wash-out.Then on 4-12% SDS-Page gel, run the IP sample, transfer to then and be used for western blot analysis on the film.Film is in order to the detection of getting off: (1) for the associating p85 of IRS-1, i.e. activatory PI3K (shown in Figure 11 A) is with the p85mAb (from Upstate, product #05-217) of 1:100 dilution; (2) for total IRS-1 (shown in Figure 11 B), with the IRS-1mAb (from BDBiosciences, product #611395) of 1:600 dilution as the loading contrast.
Data presentation IL-6 handles and to cause insulin-induced IR, the IRS-1 and the Akt phosphorylation that reduce.When with anti-IL-6 antibodies (clone AME-19a) pretreatment cell, the effect of this IL-6 has disappeared.In addition, IL-6 has suppressed the association of insulin-induced p85 (PI3K subunit) Yu IRS-1.Equally, by also suppressed the effect of this IL-6 with the anti-IL-6 antibodies pre-treatment.
Experiment in the body
In animal, also do not carry out the extensive testing of IL-6 to the insulin sensitivity effect.Whether can improve insulin sensitivity and T2DM in order to assess anti-IL-6 treatment, (obtain from R﹠amp with commercial anti-mouse IL-6 antibody; D Systems) the db/db mouse and the C57/B16 male mice of processing high fat diet.
The db/db mouse
Use the db/db mouse of different ages to test the effect that anti-IL-6 handles.The mouse in age in 8-10 week has the feature of hyperinsulinemia and insulin resistance, has therefore represented early stage disease, and 12-14 mouse in age in week also has the glucose level of rising except that hyperinsulinemia, so has represented T2DM in late period.In intraperitoneal glucose tolerance test (ipGTT), the mouse of these two age groups all is used to test the ability of anti-IL-6 treatment improvement insulin sensitivity and glycerine contrast.
Owing to have sudden change in the Leptin acceptor, so the db/db mouse has non-functional Leptin signal conduction.Along with mouse these mouse that grow up are developed into obesity, hyperinsulinemia and insulin resistance,, detect the symptom that occurs at first as mouse 6-8 during age in week.Handled with the anti-IL-6mAb of 5mg/kg the mouse-8 and 12 ages in week of two groups of different ages, and after processing, carried out intraperitoneal glucose tolerance test (ipGTT) on the the 1st and 7 day.Handling procedure as shown in Figure 15.
In 8 week age animals, glucose clearance in the GTT process is not worked with anti-IL-6mAb processing.Anti-IL-6mAb handles and causes the glucose tolerance (GT) improved in 12 week age animals, although effect is not significant (p=0.063) on the statistics.Observed this GTT and improved in the 7th day after the processing.In addition, the adipocyte factor and the adiponectin of having analyzed in the serum sample of studying before and after finishing distributes.The level of IL-6, TNF α and MCP-1 is lower than detection level.Consider these data in the lump and from the result of ipGTT, can hint: the db/db animal is not to be used to study the good model of anti-IL-6 to the insulin resistance effect; And for IL-6 possible effect of playing in insulin resistance and T2DM development, IL-6 organizes level more relevant.
The obesity of diet induced (DIO)-the be used for animal model of obesity and insulin resistance
Feed the food 20-35 week of containing 60% fat to the C57/B1 male mice.They develop into obesity (mean body weight is 50.5g) and have increased fasting blood glucose level (FBG〉145mg/dl).In addition, they have impaired GT.With 10mg/kg mouse-anti IL-6Ab (R﹠amp; DSystems) handle the DIO animal.Generally speaking, they have accepted the anti-IL-6mAb of 50mg/kg in 3 weeks.IpGTT is carried out in (the 5th day), the 4th administration (the 12nd and 16 day) back and the 5th administration (the 23rd day) after initial 2 administrations.Meanwhile, blood sampling is used for the measurement of the adipocyte factor and adiponectin.
Anti-IL-6 handled and can not improve glucose tolerance at the 5th and 12 day; Yet,, observe glucose clearance and the glucose offset level all improves when when carrying out in the 16th and 23 day.Reaching remarkable on the statistics this improvement in 39,60 and 90 minutes during the GTT.
In another group experiment, with 10 and the anti-IL-6Ab of 20mg/kg and the contrast of 20mg/kg IgG isotype by the intraperitoneal approach handle weekly the DIO animal once (the 1st all in administration 2 times, after the administration 1 time weekly of 4 weeks).Having carried out HOMA-IR (after handling for 2,4 and 6 weeks), ipGTT, ipITT and the adipocyte factor distributes (the 6th week of handling).
The DIO animal that antagonism IL-6Ab handles carries out HOMA-IR and analyzes:
In these researchs, with 10 and the DIO animal of 20mg/kg mouse-anti IL-6Ab and isotype control treatment in fasting plasma glucose and insulin level all reduced.To animal blood drawing and use trace/DMA glucose (ox) (thermo Electron Corp) respectively and hypersensitive rat insulin EIisa (Crystal Chem) mensuration fasting glucose and insulin level.These values are used to measure HOMA-IR.The HOMA-IR index has reflected the situation of insulin sensitivity, and its result with pressing plate (clamp) research is very relevant.Pass through formula: HOMA-IR (Figure 13 A, B and C) is calculated in (fasting glucose (mM) X fasting insulin (mIU/Lit))/22.5.
After handling for 2,4 and 6 weeks, observe improving (Figure 13 A-C has shown the data after handling for 6 weeks) of HOMA-IR.Studying latter stage, carry out ipGTT and ipITT.In these two tests, when comparing with the animal that isotype is handled, anti-IL-6 handles (20mg/ml) and has improved glucose skew and clearance rate significantly.
The adipocyte factor and the cytokine analysis of the serum sample of the animal of handling from contrast and anti-IL-6 show the trend that reduces in company with MCP-1 and phylaxin level, IL-6 neutralization cause the circulating minimizing of IL-6 and TNF alpha levels.In another group data, increased the adiponectin level with anti-IL-6 processing.
Carried out histologic analysis from processing and control group liver sample.Measure lipid content in the liver parenchyma with the oil red O stain sample.Descend in response to liver lipid content in the processing DIO animal of mouse-anti IL-6 antibody.
The liver sample that dyeing has disclosed in untreated animal 34% vehicle treated is that lipid is relevant, then has only 8% (Figure 14 A-F) in the animal that the anti-IL-6 of 20mg/kg handles.Figure 14 A and D are control group; Figure 14 B and E are untreated DIO animal; And Figure 14 C and F are the animal that anti-IL-6 handles.The lipid liver content that increases is relevant with the development of insulin resistance and type ii diabetes.Therefore, it is contemplated that the IL-6 neutralization causes insulin sensitivity and T2DM to improve by influencing the liver lipid metabolism.These aggregation of data get up and have hinted the effect of IL-6 in type ii diabetes pathology forcefully, and in and IL-6 can improve insulin sensitivity.
Extra research
Monitored and exist or do not exist under people's through engineering approaches anti-IL-6 antibodies, IL-6 to the IRS1 phosphorylation of insulin stimulating, the relation with p85/PI3K, insulin receptor (IR) phosphorylation, glycogen is synthetic and the effect that relates to SOCS3 and the conduction of STAT signal in the HepG2 cell.Extra experimental study the effect from islet secretion Regular Insulin of IL-6 to glucose induction.Up to now the data description of being announced IL-6 to inhibition and hormesis from the rat Langerhans islet excreting insulin.In the glucose existence or not, handle the rat Langerhans islet (from Liefscann) of fresh separated with IL-6 and people's through engineering approaches anti-IL-6 antibodies (AME-19a).Measured the insulin level of under different processing, secreting from pancreas islet.
The C2C12.C2C12 cell is used to study the effect of Regular Insulin to skeletal muscle.Experimentize to check that IRS1 and Glut4 express, insulin-induced IRS 1 phosphorylation and IL-6 are to the influence of adiponectin effect.
Advantage:
Active inhibition can have significant treatment advantage to IL-6 by IL-6 antibody of the present invention, because it can improve insulin sensitivity and metabolism is controlled and the existing Side effects of pharmaceutical drugs of nothing.In addition, the internal cause of systemic inflammatory-be considered to T2DM, relevant diabetic complication is seldom controlled in current treatment.Resemble the therapeutical agent of IL-6 antibody of the present invention, except that increasing insulin sensitivity, should be able to suppress systemic inflammatory and stop the development of diabetic complication.
Patient's quantity of suffering from T2DM constantly increases, and expects 2025 and reaches 300,000,000.Anti-IL-6 antibodies can be used as monotherapy or with other OAD that exists already, for example sulfonylurea, biguanides (as Metphormin), thiazolidinediones, meglitinide (as repaglinide), alpha-glucosidase inhibitor (as acarbose) are united use.In addition, can for example improve the control of insulin sensitivity and glucemia with Regular Insulin or other treatment agent and the therapeutical agent avoiding handling relevant hypoglycemia incident with Regular Insulin is united use.Also expected is except that improving insulin sensitivity and regulate the glucose level in T2D and metabolism syndrome patient, and anti-IL-6 treatment meeting has useful effect to the frequent observed CV change of institute in these patients.Referring to Saltiel, AR, and Kahn, CR.2001.Nature 414:799-806; Hansen, BC., 1995, Diabetes Care 18:A2-A9; Diabetes Prevention Programresearch group.2002.New Eng1.J Med., 346:393-403; Hansen, BC., 2000, AnnNew York Academy of Science, 892:1-24; Hsueh, WA., and Quinones, MJ., 2003, Am.J.Cardiology, 93:10J-17J; Resnick, HE and Howard, BV., 2002, Ann.Rev.Med., 53:245-267; Korner, J. and Aronne, L., 2003, J Clin.Invest., 111 (5): 565-570; Skoog, T., etc., 2001.Diabetologia, 44:654:655; Fernandez-Real, JM., and RicartW., 2003, Endocrine Reviews, 24 (3): 278-301; Fernandez-Real, JM., etc., 2001, JClin Endocrinol Metab., 86:1154-1159.; 10a.Fried, S., etc., 1998.J ClinEndocrinol Metab., 83:847-850; Senn, JJ., etc., 2002, Diabetes, 51:3391-3399; Rotter, V., etc., 2003, during JBC sends to press, Manus.#301977200; 12a.Stouthard, JM., etc., 1996, BBRC, 220:241-245; Southern, C., etc., 1990, Biochem J., 272:243-245; Sandler, S., etc., 1990, Endocrinology, 126:1288-1294; Pedersen, BK., etc., 2001, JPhysiol., 536:329-337; DiCosmo, BF, etc., 1994, Int.Immunol., 6:1829-1837; Wallenius, V., etc., 2002, Nature Medicine, 8:75-79; Vozarova, B., etc., 2003, Human Genetic, 112:409-413; Kubaszek, A., etc., 2003, Diabetes, 52:558-461; Tsigos, C., etc., 1997, J Clin Endocrinol Metab, 82:4167-4170; Stoutharad, JM., etc., 1995, Am J Physiol., 268; E813-E819; Kern, PA., etc., 2001, Am J PhysiolEndocrinol Metab., 280:E745-E751; Bastard, JP., etc., 2000, J Clon EndocrinolMetab., 85:3338-3342; And Bastard, JP., etc., 2002, J.Clin.Endocrinol.Metab., 87:2084-2089.
Obviously remove specification sheets before and embodiment detailed description, also can implement the present invention.Can carry out many modifications of the present invention and change according to above-mentioned instruction, and therefore fall in the scope of additional claim.
Table 1-light chain CDRs
SEQ ID NO The CDR title * The clone Sequence
SEQ ID NO：1 CDRL1 33 SASHSVSYMY
SEQ ID NO The CDR title * The clone Sequence
SEQ ID NO：2 CDRL1 33 AGTGCCAGCCATAGTGTAAGTTACATGTAC
SEQ ID NO：3 CDRL1 34 SASISVSYMY
SEQ ID NO：4 CDRL1 34 AGTGCCAGCATTAGTGTAAGTTACATGTAC
SEQ ID NO：5 CDRL1 35 SARSSVSYMY
SEQ ID NO：6 CDRL1 35 AGTGCCCGGTCAAGTGTAAGTTACATGTAC
SEQ ID NO：7 CDRL1 36 SASYSVSYMY
SEQ ID NO：8 CDRL1 36 AGTGCCAGCTATAGTGTAAGTTACATGTAC
SEQ ID NO：9 CDRL1 37 SASSSVFYMY
SEQ ID NO：10 CDRL1 37 AGTGCCAGCTCAAGTGTATTTTACATGTAC
SEQ ID NO：11 CDRL1 39 SGSSYVSYMY
SEQ ID NO：12 CDRL1 39 AGTGGCAGCTCATATGTAAGTTACATGTAC
SEQ ID NO The CDR title * The clone Sequence
SEQ ID NO：13 CDRL1 40 SALSSVSYMY
SEQ ID NO：14 CDRL1 40 AGTGCCCTGTCAAGTGTAAGTTACATGTAC
SEQ ID NO：15 CDRL1 A9 SASSSVSYMY
SEQ ID NO：16 CDRL1 A9 AGTGCCAGCTCAAGTGTAAGTTACATGTAC
SEQ ID NO：17 CDRL2 41 DFSNLAS
SEQ ID NO：18 CDRL2 41 GACTTTTCCAACCTGGCTTCT
SEQ ID NO：19 CDRL2 43 DLSNLAS
SEQ ID NO：20 CDRL2 43 GACCTGTCCAACCTGGCTTCT
SEQ ID NO：21 CDRL2 44 DMSNLAS
SEQ ID NO：22 CDRL2 44 GACATGTCCAACCTGGCTTCT
SEQ ID NO：23 CDRL2 46 DTSNLTS
SEQ ID NO The CDR title * The clone Sequence
SEQ ID NO：24 CDRL2 46 GACACATCCAACCTGACGTCT
SEQ ID NO：25 CDRL2 48 DTSELAS
SEQ ID NO：26 CDRL2 48 GACACATCCGAGCTGGCTTCT
SEQ ID NO：27 CDRL2 A9 DTSNLAS
SEQ ID NO：28 CDRL2 A9 GACACATCCAACCTGGCTTCT
SEQ ID NO：29 CDRL3 49 MQWSGYPYT
SEQ ID NO：30 CDRL3 49 ATGCAGTGGAGTGGTTACCCATACACG
SEQ ID NO：31 CDRL3 50 CQWSGYPYT
SEQ ID NO：32 CDRL3 50 TGTCAGTGGAGTGGTTACCCATACACG
SEQ ID NO：33 CDRL3 52 SCWSGYPYT
SEQ ID NO：34 CDRL3 52 TCTGTGTGGAGTGGTTACCCATACACG
SEQ ID NO The CDR title * The clone Sequence
SEQ ID NO：35 CDRL3 A9 SQWSGYPYT
SEQ ID NO：36 CDRL3 A9 TCTCAGTGGAGTGGTTACCCATACACG
SEQID NO：138 CDRL3 Alt. QQWSGYPYT
*Except that CDRH1 was Kabat and Chothia definition sum, CDRs was the Kabat definition.
Table 2-heavy chain CDRs
SEQ ID NO The CDR title * The clone Sequence
SEQ ID NO：37 CDRH1 4 GFTFSSFALS
SEQ ID NO：38 CDRH1 4 GGATTCACCTTTAGTAGCTTTGCCCTTTCT
SEQ ID NO：39 CDRH1 5 GFTFSPFAMS
SEQ ID NO：40 CDRH1 5 GGATTCACCTTTAGTCCTTTTGCCATGTCT
SEQ ID NO：41 CDRH1 6 GFQFSSFAMS
SEQ ID NO：42 CDRH1 6 GGATTCCAGTTTAGTAGCTTTGCCATGTCT
SEQ ID NO：43 CDRH1 8 GFTTSSFAMS
SEQ ID NO：44 CDRH1 8 GGATTCACCACTAGTAGCTTTGCCATGTCT
SEQ ID NO The CDR title * The clone Sequence
SEQ ID NO：45 CDRH1 Q+P GFQFSPFAMS
SEQ ID NO：46 CDRH1 Q+P GGATTCCAGTTTAGTCCTTTTGCCATGTCT
SEQ ID NO：47 CDRH1 A9 GFTFSSFAMS
SEQ ID NO：48 CDRH1 A9 GGATTCACCTTTAGTAGCTTTGCCATGTCT
SEQ ID NO：49 CDRH2 10 KASSGGSYTYYPDTVTG
SEQ ID NO：50 CDRH2 10 AAAGCGAGTAGTGGTGGGAGTTACACCTACTATCCTGA CACTGTGACGGGC
SEQ ID NO：51 CDRH2 11 KISSGGSYEYYPDTVTG
SEQ ID NO：52 CDRH2 11 AAAATTAGTAGTGGTGGGAGTTACGAGTACTATCCTGA CACTGTGACGGGC
SEQ ID NO：53 CDRH2 12 KISSGGSYYYYPDTVTG
SEQ ID NO：54 CDRH2 12 AAAATTAGTAGTGGTGGGAGTTACTATTACTATCCTGA CACTGTGACGGGC
SEQ ID NO：55 CDRH2 14 KISSGGSWTYYPDTVTG
SEQ ID NO：56 CDRH2 14 AAAATTAGTAGTGGTGGGAGTTGGACCTACTATCCTGA CACTGTGACGGGC
SEQ ID NO：57 CDRH2 16 KISPGGSYTYYPDTVTG
SEQ ID NO：58 CDRH2 16 AAAATTAGTCCGGGTGGGAGTTACACCTACTATCCTGA CACTGTGACGGGC
SEQ ID NO The CDR title * The clone Sequence
SEQ ID NO：59 CDRH2 P+W +S (18a， 19a) KISPGGSWTYYSDTVTG
SEQ ID NO：60 CDRH2 P+W +S (18a， 19a) AAAATTAGTCCGGGTGGGAGTTGGACCTACTATTCTGA CACTGTGACGGGC
SEQ ID NO：61 CDRH2 A9 KISSGGSYTYYPDTVTG
SEQ ID NO：62 CDRH2 A9 AAAATTAGTAGTGGTGGGAGTTACACCTACTATCCTGA CACTGTGACGGGC
SEQ ID NO：113 CDRH2 Alt. EISSGGSYTYYPDTVTG
SEQ ID NO：63 CDRH2 17 KISSGGSYTYFPDTVTG
SEQ ID NO：64 CDRH2 17 AAAATTAGTAGTGGTGGGAGTTACACCTACTTTCCTGA CACTGTGACGGGC
SEQ ID NO：65 CDRH2 19 KISSGGSYTYYPDTVAG
SEQ ID NO：66 CDRH2 19 AAAATTAGTAGTGGTGGGAGTTACACCTACTATCCTGA CACTGTGGCTGGC
SEQ ID NO：67 CDRH2 20 KISSGGSYTYYDDTVTG
SEQ ID NO：68 CDRH2 20 AAAATTAGTAGTGGTGGGAGTTACACCTACTATGATGA CACTGTGACGGGC
SEQ ID NO：69 CDRH2 21 KISSGGSYTYYSDTVTG
SEQ ID NO The CDR title * The clone Sequence
SEQ ID NO：70 CDRH2 21 AAAATTAGTAGTGGTGGGAGTTACACCTACTATTCTGA CACTGTGACGGGC
SEQ ID NO：71 CDRH2 22 KISSGGSYTYYPDTVTP
SEQ ID NO：72 CDRH2 22 AAAATTAGTAGTGGTGGGAGTTACACCTACTATCCTGA CACTGTGACGCCG
SEQ ID NO：73 CDRH2 23 KISSGGSYTYYPDTDTG
SEQ ID NO：74 CDRH2 23 AAAATTAGTAGTGGTGGGAGTTACACCTACTATCCTGA CACTGATACGGGC
SEQ ID NO：75 CDRH2 P+S (20b， 23a) KISPGGSYTYYSDTVTG
SEQ ID NO：76 CDRH2 P+S (20b， 23a) AAAATTAGTCCGGGTGGGAGTTACACCTACTATTCTGA CACTGTGACGGGC
SEQ ID NO：77 CDRH2 P+W +D (22a) KISPGGSWTYYDDTVTG
SEQ ID NO：78 CDRH2 P+W +D (22a) AAAATTAGTCCGGGTGGGAGTTGGACCTACTATGATGA CACTGTGACGGGC
SEQ ID NO：79 CDRH3 25 QLWGSYALDY
SEQ ID NO：80 CDRH3 25 CAGTTATGGGGGTCGTATGCTCTTGACTAC
SEQ ID NO：81 CDRH3 26 QLWGYYALDT
SEQ ID NO The CDR title * The clone Sequence
SEQ ID NO：82 CDRH3 26 CAGTTATGGGGGTACTATGCTCTTGACACG
SEQ ID NO：83 CDRH3 29 QLWGTYALDY
SEQ ID NO：84 CDRH3 29 CAGTTATGGGGGACTTATGCTCTTGACTAC
SEQ ID NO：85 CDRH3 30 QLWGNYALDY
SEQ ID NO：86 CDRH3 30 CAGTTATGGGGGAATTATGCTCTTGACTAC
SEQ ID NO：87 CDRH3 31 QLWGYYALDF
SEQ ID NO：88 CDRH3 31 CAGTTATGGGGGTACTATGCTCTTGACTTT
SEQ ID NO：89 CDRH3 32 QLWGYYALDI
SEQ ID NO：90 CDRH3 32 CAGTTATGGGGGTACTATGCTCTTGACATT
SEQ ID NO：91 CDRH3 A9 QLWGYYALDY
SEQ ID NO：92 CDRH3 A9 CAGTTATGGGGGTACTATGCTCTTGACTAC
SEQ ID NO：114 CDRH3 Alt. GLWGYYALDY
*Except that CDRH1 was Kabat and Chothia definition sum, CDRs was the Kabat definition.
Table 3-is from the sudden change in individual CDR library
The clone CDRH1 The clone CDRL1
4 M34L 33 S27H
5 S31P 34 S271
6 T28Q 35 S26R
8 F29T 36 S27Y
37 S30F
The clone CDRH2 38 S271
10 151A 39 A25G，S28Y
11 T57E 40 S26L
12 T57Y
14 Y56W The clone CDRL2
16 S52aP 41 T51F
17 Y59F 43 T51L
19 T64A 44 T51M
20 P60D 46 A55T
21 P60S 47 T51L
22 G65P 48 N53E
23 V63D
The clone CDRL3
The clone CDRH3 49 Q89M
25 Y99S 50 Q89C
26 Y102T 52 Q90C
27 Y99S
29 Y99T
30 Y99N
31 Y102F
32 Y1021
Table 4-is included in the sudden change in the combinatorial library
CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3
T28Q S52aP Y102F S271 T51F Q89M
S31P Y56W Y1021 S27Y T51M
P60S
V63D
Table 5A
Positive library clone
Figure A200680024037D01171
Table 5B-people through engineering approaches anti-IL-6 antibodies clone and corresponding C DRs
CDR--> L1 L2 L3 H1 H2 H3
AME-A9 SEQ ID：15 SEQ ID：27 SEQ ID：35 SEQ ID：47 SEQ ID：61 SEQ ID：91
AME-16 SEQ ID：15 SEQ ID：27 SEQ ID：35 SEQ ID：47 SEQ ID：57 SEQ ID：91
AME-18a SEQ ID：15 SEQ ID：17 SEQ ID：29 SEQ ID：45 SEQ ID：59 SEQ ID：89
AME-19a SEQ ID：3 SEQ ID：21 SEQ ID：29 SEQ ID：39 SEQ ID：59 SEQ ID：89
AME-20b SEQ ID：3 SEQ ID：21 SEQ ID：29 SEQ ID：41 SEQ ID：75 SEQ ID：89
AME-22a SEQ ID：7 SEQ ID：17 SEQ ID：29 SEQ ID：45 SEQ ID：77 SEQ ID：87
AME-23a SEQ ID：7 SEQ ID：21 SEQ ID：29 SEQ ID：41 SEQ ID：75 SEQ ID：87
Table 6-EC 50Value
The clone The EC50 value
CNTO328 2.7 x 10 -11M
AME-19a 2.7 x 10 -12M (10-doubly improves)
The kinetic constant of the anti-IL-6IgG of table 7-
The clone Antibody concentration (pM) K D(pM) Improve ratio (comparing) with chimeric ab kon (M -1sec -1) Improve ratio k off(sec -1) (calculating) Improve ratio
Chimeric antibody 5 3 1 4.4 x 10 6 1 1.3 x 10 -5 1
AME-16 1 0.83 3.6 1 x 10 6 0.22 8.3 x 10 -7 15.7
AME-18a 0.5 0.12 25 2 x 10 7 4.4 2.4 x 10 -6 5.4
AME-19a 0.5 0.037 81.1 5.5 x 10 6 1.2 2 x 10 -7 65
AME-20b 1 0.78 3.8 4.7 x 10 6 1 3.7 x 10 -6 3.5
AME-22a 1 0.18 16.7 6 x 10 6 1.3 1.1 x 10 -6 11.8
AME-23a 1 0.006 500 7.4 x 10 6 1.6 4.4 x 10 -8 295
The cross species reactivity of table 8-people's engineering and chimeric antibody
Figure A200680024037D01201
The cross species reactivity of people's engineering and chimeric antibody.The 7TD1 cell proliferation that the neutralization of people's engineering and inosculating antibody physical efficiency is stimulated for the condition supernatant liquor from the PBMCs of people, marmoset monkey (Marmoset), rhesus monkey (Cynomolgous), chimpanzee (Chimp), macaque (Rhesus), baboon (Baboon), stump-tailed macaque (Pig-Tail) and thin,tough silk hair monkey (Cotton Top)."+" positive in neutralization is measured; "-" feminine gender in neutralization is measured; N/D, undetermined.
The anti-IL-6mAb of table 9-handles the pathological influence of NZB/W F1 mouse middle kidney
*Around severe-blood vessel Combination lymphoidocyte hyperplasia, mesangial cell too much, proteinosis, glomerular basement membrane immune complex deposit
Around moderate-moderate blood vessel Combination lymphoidocyte hyperplasia, moderate mesangial cell too much, glomerular basement membrane immune complex deposit, no proteinosis
Slightly-slight mesangial cell too much, slight glomerular basement membrane immune complex deposit, no blood vessel Combination lymphoidocyte hyperplasia, no proteinosis on every side
Treatment group Severe * Moderate * Slightly *
Salt solution (n=10) 60% or 6/10 20% or 2/10 20% or 2/10
Rat IgG (n=10) 70% or 7/10 30% or 3/10 0 or 0/10
The anti-mouse IL-6 of R﹠D (n=10) 10% or 1/10 30% or 3/10 60% or 6/10
Table 10-clone's variable region sequences
SEQ ID NO The clone Heavy (H) or light (L) chain V district Sequence
93 A9 L chain AA EIVLTQSPATLSLSPGERATLSCSASSSVS YMYWYQQKPGQAPRLLIYDTSNLASGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCSQW SGYPYTFGGGTKVEIK
94 L chain Nucleotide GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA AAGAGCCACCCTCTCCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACT GGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGACACA TCCAACCTGGCTTCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGG GACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAG TTTATTACTGTTCTCAGTGGAGTGGTTACCCATACACGTTCGGCGGAGGG ACCAAGGTGGAGATCAAA
95 H chain AA EVQLVESGGGLVQPGGSLRLSCAASGFTFS SFAMSWVRQAPGKGLEWVAKISSGGSYTYY PDTVTGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARQLWGYYALDYWGQGTTVTVSS
96 H chain Nucleotide GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTTTGCCA TGTCTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAA ATTAGTAGTGGTGGGAGTTACACCTACTATCCTGACACTGTGACGGGCCG ATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGA ACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACAGTTA TGGGGGTACTATGCTCTTGACTACTGGGGCCAAGGGACCACGGTCACCGT CTCCTCA
97 19A L chain AA EIVLTQSPATLSLSPGERATLSCSASISVS YMYWYQQKPGQAPRLLIYDMSNLASGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCMQW SGYPYTFGGGTKVEIK
98 L chain Nucleotide GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA AAGAGCCACCCTCTCCTGCAGTGCCAGCATTAGTGTAAGTTACATGTACT GGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGACATG TCCAACCTGGCTTCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGG GACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAG TTTATTACTGTATGCAGTGGAGTGGTTACCCATACACGTTCGGCGGAGGG ACCAAGGTGGAGATCAAA
99 H chain AA EVQLVESGGGLVQPGGSLRLSCAASGFTFS PFAMSWVRQAPGKGLEWVAKISPGGSWTYY SDTVTGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARQLWGYYALDIWGQGTTVTVSS
100 H chain Nucleotide GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTCCTTTTGCCA TGTCTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAA ATTAGTCCGGGTGGGAGTTGGACCTACTATTCTGACACTGTGACGGGCCG ATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGA ACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACAGTTA TGGGGGTACTATGCTCTTGACATTTGGGGCCAAGGGACCACGGTCACCGT CTCCTCA
101 23A L chain AA EIVLTQSPATLSLSPGERATLSCSASYSVS YMYWYQQKPGQAPRLLIYDMSNLASGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCMQW SGYPYTFGGGTKVEIK
102 L chain Nucleotide GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA AAGAGCCACCCTCTCCTGCAGTGCCAGCTATAGTGTAAGTTACATGTACT GGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGACATG TCCAACCTGGCTTCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGG GACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAG TTTATTACTGTATGCAGTGGAGTGGTTACCCATACACGTTCGGCGGAGGG ACCAAGGTGGAGATCAAA
103 H chain AA EVQLVESGGGLVQPGGSLRLSCAASGFQFS SFANSWVRQAPGKGLEWVAKISPGGSYTYY SDTVTGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARQLWGYYALDFWGQGTTVTVSS
104 H chain Nucleotide GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC CCTGAGACTCTCCTGTGCAGCCTCTGGATTCCAGTTTAGTAGCTTTGCCA TGTCTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAA ATTAGTCCGGGTGGGAGTTACACCTACTATTCTGACACTGTGACGGGCCG ATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGA ACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACAGTTA TGGGGGTACTATGCTCTTGACTTTTGGGGCCAAGGGACCACGGTCACCGT CTCCTCA
116 AME-16 L chain AA EIVLTQSPATLSLSPGERATLSCSASSSVS YMYWYQQKPGQAPRLLIYDTSNLASGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCSQW SGYPYTFGGGTKVEIK
117 L chain Nucleotide ATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGA TACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGTGCCAGCTCAAGTGTAAGT TACATGTACTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCAT CTATGACACATCCAACCTGGCTTCTGGCATCCCAGCCAGGTTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAA GATTTTGCAGTTTATTACTGTTCTCAGTGGAGTGGTTACCCATACACGTT CGGCGGAGGGACCAAGGTGGAGATCAAA
118 H chain AA EVQLVESGGGLVQPGGSLRLSCAASGFTFS SFAMSWVRQAPGKGLEWVAKISPGGSYTYY PDTVTGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARQLWGYYALDYWGQGTTVTVSS
119 H chain Nucleotide ATGGAGTTTGGCCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAAGGTGT CCAGTGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTG GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGC TTTGCCATGTCTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGT GGCCAAAATTAGTCCCGGTGGGAGTTACACCTACTATCCTGACACTGTGA CGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAG ACAGTTATGGGGGTACTATGCTCTTGACTACTGGGGCCAAGGGACCACGG TCACCGTCTCCTCA
120 AME-18a L chain AA EIVLTQSPATLSLSPGERATLSCSASSSVS YMYWYQQKPGQAPRLLIYDFSNLASGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCMQW SGYPYTFGGGTKVEIK
121 L chain Nucleotide ATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGA TACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGTGCCAGCTCAAGTGTAAGT TACATGTACTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCAT CTATGACTTCTCCAACCTGGCTTCTGGCATCCCAGCCAGGTTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAA GATTTTGCAGTTTATTACTGTATGCAGTGGAGTGGTTACCCATACACGTT CGGCGGAGGGACCAAGGTGGAGATCAAA
122 H chain AA EVQLVESGGGLVQPGGSLRLSCAASGFQFS PFAMSWVRQAPGKGLEWVAKISPGGSWTYY SDTVTGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARQLWGYYALDIWGQGTTVTVSS
123 H chain Nucleotide ATGGAGTTTGGCCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAAGGTGT CCAGTGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTG GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCCAGTTTAGTCCC TTTGCCATGTCTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGT GGCCAAAATTAGTCCCGGTGGGAGTTGGACCTACTATAGCGACACTGTGA CGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAG ACAGTTATGGGGGTACTATGCTCTTGACATTTGGGGCCAAGGGACCACGG TCACCGTCTCCTCA
124 AME-20b L chain AA EIVLTQSPATLSLSPGERATLSCSASISVS YMYWYQQXPGQAPRLLIYDMSNLASGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCMQW SGYPYTFGGGTKVEIK
125 L chain Nucleotide ATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGA TACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGTGCCAGCATTAGTGTAAGT TACATGTACTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCAT CTATGACATGTCCAACCTGGCTTCTGGCATCCCAGCCAGGTTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAA GATTTTGCAGTTTATTACTGTATGCAGTGGAGTGGTTACCCATACACGTT CGGCGGAGGGACCAAGGTGGAGATCAAA
126 H chain AA EVQLVESGGGLVQPGGSLRLSCAASGFQFS SFAMSWVRQAPGKGLEWVAKISPGGSYTYY SDTVTGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARQLWGYYALDIWGQGTTVTVSS
127 H chain Nucleotide ATGGAGTTTGGCCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAAGGTGT CCAGTGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTG GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCCAGTTTAGTAGC TTTGCCATGTCTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGT GGCCAAAATTAGTCCCGGTGGGAGTTACACCTACTATAGCGACACTGTGA CGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAG ACAGTTATGGGGGTACTATGCTCTTGACATTTGGGGCCAAGGGACCACGG TCACCGTCTCCTCA
128 AME-22a L chain AA EIVLTQSFATLSLSPGERATLSCSASYSVS YMYWYQQKPGQAPRLLIYDFSNLASGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYCMQW SGYPYTFGGGTKVEIK
129 L chain Nucleotide ATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGA TACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGT CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGTGCCAGCTACAGTGTAAGT TACATGTACTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCAT CTATGACTTCTCCAACCTGGCTTCTGGCATCCCAGCCAGGTTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAA GATTTTGCAGTTTATTACTGTATGCAGTGGAGTGGTTACCCATACACGTT CGGCGGAGGGACCAAGGTGGAGATCAAA
130 H chain AA EVQLVESGGGLVQPGGSLRLSCAASGFQFS PFAMSWVRQAPGKGLEWVAKISPGGSWTYY PDTDTGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARQLWGYYALDFWGQGTTVTVSS
131 H chain Nucleotide ATGGAGTTTGGCCTGAGCTGGGTTTTCCTTGTTGCTATTTTAGAAGGTGT CCAGTGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTG GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCCAGTTTAGTCCC TTTGCCATGTCTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGT GGCCAAAATTAGTCCCGGTGGGAGTTGGACCTACTATCCTGACACTGACA CGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAG ACAGTTATGGGGGTACTATGCTCTTGACTTCTGGGGCCAAGGGACCACGG TCACCGTCTCCTCA
The aminoacid sequence of people's light chain framework region L6 that table 11-has the CDR sequence that is interspersed of mark
(FRL1-SEQ ID NO：105) CDRL1 (FRL2-SEQ ID NO：106) CDRL2
EIVLTQSPATLSLSPGERATLSC XXXXXXXXXXWYQQKPGQAPRLLIY XXXXXXX
(FRL3-SEQ ID NO：107) CDRL3(FRL4-SEQ ID NO：108)
GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC XXXXXXXXXFGGGTKVEIK
The aminoacid sequence of people's heavy chain framework region VH3-7 that table 12-has the CDR sequence that is interspersed of mark
(FRH1-SEQ ID NO：109) CDRH1 (FRH2-SEQ ID NO：110)
EVQLVESGGGLVQPGGSLRLSCAAS XXXXXXXXXXWVRQAPGKGLEWVA
CDRH2 (FRH3-SEQ ID NO：111)
XXXXXXXXXXXXXXXXXRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
CDRH3 (FRH4-SEQ ID NO：112)
XXXXXXXXXXWGQGTTVTVSS
Table 13-CDR sequence
SEQ ID NO： CDR The AA sequence *
132 CDRL1 S X 1 X 2 X 3 X 4 V X 5 YMY
133 CDRL2 D X 6 S X 7 L X 8 S
134 CDRL3 X 9 X 10 WSGYPYT
135 CDRH1 GF X 11 X 12 S X 13 FA X 14 S
136 CDRH2 K X 15 S X 16 GGS X 17 X 18 Y X 19 X 20 DT X 21 X 22 X 23
137 CDRH3 QLWG X 24 YALD X 25
*X represent any have be shown in table 1 and 2 and the SEQ ID NOS:1-92 of table 3,4,5A and 8 disclosed in sequence in the amino acid that is fit to illustration, unrestriced aminoacid replacement.
In addition, X can have following train value:
X 1=A or G
X 2=S or R
X 3=H, I, S or Y
X 4=S or Y
X 5=S or F
X 6=F, L, M or T
X 7=N or E
X 8=A or T
X 9=M, C or S
X 10=Q or C
X 11=T or Q
X 12=F, S or T
X 13=S or P
X 14=L or M
X 15=A or I
X 16=S or P
X 17=Y or W
X 18=T, E or Y
X 19=Y or F
X 20=P, S, D or F
X 21=V or D
X 22=T or A
X 23=G or P
X 24=S, Y, T or N
X 25=Y, T, F or I
The proteic aminoacid sequence of SEO ID NO:115-IL-6
MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERIDKQ
IRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCL
VKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPD
PTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM
Sequence table
<110>Centocor，Inc.
Applied Molecular Evolution，Ino.
＜120〉anti--IL-6 antibody, composition, method and application
<130>CEN5094 PCT
<140>PCT/US2006/016457
<141>2006-04-28
<150>60/676,498
<151>2005-04-29
<150>60/677,319
<151>2005-05-03
<160>138
<170>FastSEQ for Windows Veraion 4.0
<210>1
<211>10
<212>PRT
＜213〉homo sapiens
<400>1
Figure A200680024037Q01291
<210>2
<211>30
<212>DNA
＜213〉homo sapiens
<400>2
Figure A200680024037Q01292
<210>3
<211>10
<212>PRT
＜213〉homo sapiens
<400>3
Figure A200680024037Q01293
<210>4
<211>30
<212>DNA
＜213〉homo sapiens
<400>4
Figure A200680024037Q01294
<210>5
<211>10
<212>PRT
＜213〉homo sapiens
<400>5
<210>6
<211>30
<212>DNA
＜213〉homo sapiens
<400>6
<210>7
<211>10
<212>PRT
＜213〉homo sapiens
<400>7
<210>8
<211>30
<212>DNA
＜213〉homo sapiens
<400>8
Figure A200680024037Q01304
<210>9
<211>10
<212>PRT
＜213〉homo sapiens
<400>9
Figure A200680024037Q01305
<210>10
<211>30
<212>DNA
＜213〉homo sapiens
<400>10
Figure A200680024037Q01306
<210>11
<211>10
<212>PRT
＜213〉homo sapiens
<400>11
<210>12
<211>30
<212>DNA
＜213〉homo sapiens
<400>12
Figure A200680024037Q01311
<210>13
<211>10
<212>PRT
＜213〉homo sapiens
<400>13
Figure A200680024037Q01312
<210>14
<211>30
<212>DNA
＜213〉homo sapiens
<400>14
Figure A200680024037Q01313
<210>15
<211>10
<212>PRT
＜213〉homo sapiens
<400>15
Figure A200680024037Q01314
<210>16
<211>30
<212>DNA
＜213〉homo sapiens
<400>16
<210>17
<211>7
<212>PRT
＜213〉homo sapiens
<400>17
Figure A200680024037Q01316
<210>18
<211>21
<212>DNA
＜213〉homo sapiens
<400>18
<210>19
<211>7
<212>PRT
＜213〉homo sapiens
<400>19
Figure A200680024037Q01322
<210>20
<211>21
<212>DNA
＜213〉homo sapiens
<400>20
Figure A200680024037Q01323
<210>21
<211>7
<212>PRT
＜213〉homo sapiens
<400>21
Figure A200680024037Q01324
<210>22
<211>21
<212>DNA
＜213〉homo sapiens
<400>22
Figure A200680024037Q01325
<210>23
<211>7
<212>PRT
＜213〉homo sapiens
<400>23
Figure A200680024037Q01326
<210>24
<211>21
<212>DNA
＜213〉homo sapiens
<400>24
Figure A200680024037Q01327
<210>25
<211>7
<212>PRT
＜213〉homo sapiens
<400>25
Figure A200680024037Q01331
<210>26
<211>21
<212>DNA
＜213〉homo sapiens
<400>26
Figure A200680024037Q01332
<210>27
<211>7
<212>PRT
＜213〉homo sapiens
<400>27
Figure A200680024037Q01333
<210>28
<211>21
<212>DNA
＜213〉homo sapiens
<400>28
Figure A200680024037Q01334
<210>29
<211>9
<212>PRT
＜213〉homo sapiens
<400>29
Figure A200680024037Q01335
<210>30
<211>27
<212>DNA
＜213〉homo sapiens
<400>30
Figure A200680024037Q01336
<210>31
<211>9
<212>PRT
＜213〉homo sapiens
<400>31
<210>32
<211>27
<212>DNA
＜213〉homo sapiens
<400>32
Figure A200680024037Q01341
<210>33
<211>9
<212>PRT
＜213〉homo sapiens
<400>33
Figure A200680024037Q01342
<210>34
<211>27
<212>DNA
＜213〉homo sapiens
<400>34
Figure A200680024037Q01343
<210>35
<211>9
<212>PRT
＜213〉homo sapiens
<400>35
Figure A200680024037Q01344
<210>36
<211>27
<212>DNA
＜213〉homo sapiens
<400>36
Figure A200680024037Q01345
<210>37
<211>10
<212>PRT
＜213〉homo sapiens
<400>37
Figure A200680024037Q01346
<210>38
<211>30
<212>DNA
＜213〉homo sapiens
<400>38
Figure A200680024037Q01347
<210>39
<211>10
<212>PRT
＜213〉homo sapiens
<400>39
Figure A200680024037Q01351
<210>40
<211>30
<212>DNA
＜213〉homo sapiens
<400>40
<210>41
<211>10
<212>PRT
＜213〉homo sapiens
<400>41
<210>42
<211>30
<212>DNA
＜213〉homo sapiens
<400>42
Figure A200680024037Q01354
<210>43
<211>10
<212>PRT
＜213〉homo sapiens
<400>43
Figure A200680024037Q01355
<210>44
<211>30
<212>DNA
＜213〉homo sapiens
<400>44
Figure A200680024037Q01356
<210>45
<211>10
<212>PRT
＜213〉homo sapiens
<400>45
Figure A200680024037Q01361
<210>46
<211>30
<212>DNA
＜213〉homo sapiens
<400>46
Figure A200680024037Q01362
<210>47
<211>10
<212>PRT
＜213〉homo sapiens
<400>47
<210>48
<211>30
<212>DNA
＜213〉homo sapiens
<400>48
Figure A200680024037Q01364
<210>49
<211>17
<212>PRT
＜213〉homo sapiens
<400>49
Figure A200680024037Q01365
<210>50
<211>51
<212>DNA
＜213〉homo sapiens
<400>50
Figure A200680024037Q01366
<210>51
<211>17
<212>PRT
＜213〉homo sapiens
<400>51
Figure A200680024037Q01367
<210>52
<211>51
<212>DNA
＜213〉homo sapiens
<400>52
Figure A200680024037Q01371
<210>53
<211>17
<212>PRT
＜213〉homo sapiens
<400>53
Figure A200680024037Q01372
<210>54
<211>51
<212>DNA
＜213〉homo sapiens
<400>54
<210>55
<211>17
<212>PRT
＜213〉homo sapiens
<400>55
Figure A200680024037Q01374
<210>56
<211>51
<212>DNA
＜213〉homo sapiens
<400>56
<210>57
<211>17
<212>PRT
＜213〉homo sapiens
<400>57
Figure A200680024037Q01376
<210>58
<211>51
<212>DNA
＜213〉homo sapiens
<400>58
Figure A200680024037Q01381
<210>59
<211>17
<212>PRT
＜213〉homo sapiens
<400>59
Figure A200680024037Q01382
<210>60
<211>51
<212>DNA
＜213〉homo sapiens
<400>60
Figure A200680024037Q01383
<210>61
<211>17
<212>PRT
＜213〉homo sapiens
<400>61
<210>62
<211>51
<212>DNA
＜213〉homo sapiens
<400>62
Figure A200680024037Q01385
<210>63
<211>17
<212>PRT
＜213〉homo sapiens
<400>63
Figure A200680024037Q01386
<210>64
<211>51
<212>DNA
＜213〉homo sapiens
<400>64
Figure A200680024037Q01391
<210>65
<211>17
<212>PRT
＜213〉homo sapiens
<400>65
Figure A200680024037Q01392
<210>66
<211>51
<212>DNA
＜213〉homo sapiens
<400>66
Figure A200680024037Q01393
<210>67
<211>17
<212>PRT
＜213〉homo sapiens
<400>67
Figure A200680024037Q01394
<210>68
<211>51
<212>DNA
＜213〉homo sapiens
<400>68
<210>69
<211>17
<212>PRT
＜213〉homo sapiens
<400>69
Figure A200680024037Q01396
<210>70
<211>51
<212>DNA
＜213〉homo sapiens
<400>70
<210>71
<211>17
<212>PRT
＜213〉homo sapiens
<400>71
Figure A200680024037Q01402
<210>72
<211>51
<212>DNA
＜213〉homo sapiens
<400>72
Figure A200680024037Q01403
<210>73
<211>17
<212>PRT
＜213〉homo sapiens
<400>73
Figure A200680024037Q01404
<210>74
<211>51
<212>DNA
＜213〉homo sapiens
<400>74
<210>75
<211>17
<212>PRT
＜213〉homo sapiens
<400>75
Figure A200680024037Q01406
<210>76
<211>51
<212>DNA
＜213〉homo sapiens
<400>76
Figure A200680024037Q01411
<210>77
<211>17
<212>PRT
＜213〉homo sapiens
<400>77
<210>78
<211>51
<212>DNA
＜213〉homo sapiens
<400>78
Figure A200680024037Q01413
<210>79
<211>10
<212>PRT
＜213〉homo sapiens
<400>79
Figure A200680024037Q01414
<210>90
<211>30
<212>DNA
＜213〉homo sapiens
<400>80
Figure A200680024037Q01415
<210>81
<211>10
<212>PRT
＜213〉homo sapiens
<400>81
Figure A200680024037Q01416
<210>82
<211>30
<212>DNA
＜213〉homo sapiens
<400>82
Figure A200680024037Q01417
<210>83
<211>10
<212>PRT
＜213〉homo sapiens
<400>83
Figure A200680024037Q01421
<210>84
<211>30
<212>DNA
＜213〉homo sapiens
<400>84
Figure A200680024037Q01422
<210>85
<211>10
<212>PRT
＜213〉homo sapiens
<400>85
Figure A200680024037Q01423
<210>86
<211>30
<212>DNA
＜213〉homo sapiens
<400>86
Figure A200680024037Q01424
<210>87
<211>10
<212>PRT
＜213〉homo sapiens
<400>87
Figure A200680024037Q01425
<210>88
<211>30
<212>DNA
＜213〉homo sapiens
<400>88
Figure A200680024037Q01426
<210>89
<211>10
<212>PRT
＜213〉homo sapiens
<400>89
Figure A200680024037Q01431
<210>90
<211>30
<212>DNA
＜213〉homo sapiens
<400>90
Figure A200680024037Q01432
<210>91
<211>10
<212>PRT
＜213〉homo sapiens
<400>91
Figure A200680024037Q01433
<210>92
<211>30
<212>DNA
＜213〉homo sapiens
<400>92
Figure A200680024037Q01434
<210>93
<211>106
<212>PRT
＜213〉homo sapiens
<400>93
<210>94
<211>318
<212>DNA
＜213〉homo sapiens
<400>94
<210>95
<211>119
<212>PRT
＜213〉homo sapiens
<400>95
Figure A200680024037Q01442
<210>96
<211>357
<212>DNA
＜213〉homo sapiens
<400>96
<210>97
<211>106
<212>PRT
＜213〉homo sapiens
<400>97
Figure A200680024037Q01444
Figure A200680024037Q01451
<210>98
<211>318
<212>DNA
＜213〉homo sapiens
<400>98
Figure A200680024037Q01452
<210>99
<211>119
<212>PRT
＜213〉homo sapiens
<400>99
Figure A200680024037Q01453
<210>100
<211>357
<212>DNA
＜213〉homo sapiens
<400>100
Figure A200680024037Q01454
<210>101
<211>106
<212>PRT
＜213〉homo sapiens
<400>101
Figure A200680024037Q01461
<210>102
<211>318
<212>DNA
＜213〉homo sapiens
<400>102
Figure A200680024037Q01462
<210>103
<211>119
<212>PRT
＜213〉homo sapiens
<400>103
<210>104
<211>357
<212>DNA
＜213〉homo sapiens
<400>104
Figure A200680024037Q01471
<210>105
<211>23
<212>PRT
＜213〉homo sapiens
<400>105
Figure A200680024037Q01472
<210>106
<211>15
<212>PRT
＜213〉homo sapiens
<400>106
Figure A200680024037Q01473
<210>107
<211>32
<212>PRT
＜213〉homo sapiens
<400>107
Figure A200680024037Q01474
<210>108
<211>10
<212>PRT
＜213〉homo sapiens
<400>108
Figure A200680024037Q01475
<210>109
<211>25
<212>PRT
＜213〉homo sapiens
<400>109
Figure A200680024037Q01476
<210>110
<211>14
<212>PRT
＜213〉homo sapiens
<400>110
Figure A200680024037Q01481
<210>111
<211>32
<212>PRT
＜213〉homo sapiens
<400>111
Figure A200680024037Q01482
<210>112
<211>11
<212>PRT
＜213〉homo sapiens
<400>112
Figure A200680024037Q01483
<210>113
<211>17
<212>PRT
＜213〉homo sapiens
<400>113
Figure A200680024037Q01484
<210>114
<211>10
<212>PRT
＜213〉homo sapiens
<400>114
Figure A200680024037Q01485
<210>115
<211>212
<212>PRT
＜213〉homo sapiens
<400>115
Figure A200680024037Q01491
<210>116
<211>106
<212>PRT
＜213〉homo sapiens
<400>116
Figure A200680024037Q01492
<210>117
<211>378
<212>DNA
＜213〉homo sapiens
<400>117
Figure A200680024037Q01501
<210>118
<211>119
<212>PRT
＜213〉homo sapiens
<400>118
Figure A200680024037Q01502
<210>119
<211>414
<212>DNA
＜213〉homo sapiens
<400>119
Figure A200680024037Q01503
<210>120
<211>106
<212>PRT
＜213〉homo sapiens
<400>120
Figure A200680024037Q01504
Figure A200680024037Q01511
<210>121
<211>378
<212>DNA
＜213〉homo sapiens
<400>121
Figure A200680024037Q01512
<210>122
<211>119
<212>PRT
＜213〉homo sapiens
<400>122
Figure A200680024037Q01513
<210>123
<211>414
<212>DNA
＜213〉homo sapiens
<400>123
Figure A200680024037Q01514
<210>124
<211>106
<212>PRT
＜213〉homo sapiens
<400>124
Figure A200680024037Q01521
<210>125
<211>378
<212>DNA
＜213〉homo sapiens
<400>125
<210>126
<211>119
<212>PRT
＜213〉homo sapiens
<400>126
Figure A200680024037Q01523
<210>127
<211>414
<212>DNA
＜213〉homo sapiens
<400>127
Figure A200680024037Q01531
<210>128
<211>106
<212>PRT
＜213〉homo sapiens
<400>128
Figure A200680024037Q01532
<210>129
<211>378
<212>DNA
＜213〉homo sapiens
<400>129
Figure A200680024037Q01533
<210>130
<211>119
<212>PRT
＜213〉homo sapiens
<400>130
Figure A200680024037Q01541
<210>131
<211>414
<212>DNA
＜213〉homo sapiens
<400>131
Figure A200680024037Q01542
<210>132
<211>10
<212>PRT
＜213〉homo sapiens
<220>
<221>unsure
<222>(2)(3)(4)(5)(7)
＜223〉wherein, Xaa can be any one in the amino acid in naturally occurring 20.
<400>132
<210>133
<211>7
<212>PRT
＜213〉homo sapiens
<220>
<221>unsure
<222>(2)(4)(6)
＜223〉wherein, Xaa can be any one in the amino acid in naturally occurring 20.
<400>133
Figure A200680024037Q01544
<210>134
<211>9
<212>PRT
＜213〉homo sapiens
<220>
<221>unsure
<222>(1)(2)
＜223〉wherein, Xaa can be any one in the amino acid in naturally occurring 20.
<400>134
Figure A200680024037Q01551
<210>135
<211>10
<212>PRT
＜213〉homo sapiens
<220>
<221>unBure
<222>(3)(4)(6)(9)
＜223〉wherein, Xaa can be any one in the amino acid in naturally occurring 20.
<400>135
Figure A200680024037Q01552
<210>136
<211>17
<212>PRT
＜213〉homo sapiens
<220>
<221>unBure
<222>(2)(4)(8)(9)(11)(12)(15)(16)(17)
＜223〉wherein, Xaa can be any one in the amino acid in naturally occurring 20.
<400>136
Figure A200680024037Q01553
<210>137
<211>10
<212>PRT
＜213〉homo sapiens
<220>
<221>unsure
<222>(5)(10)
＜223〉wherein, Xaa can be any one in the amino acid in naturally occurring 20.
<400>137
Figure A200680024037Q01561
<210>138
<211>9
<212>PRT
＜213〉homo sapiens
<400>138
Figure A200680024037Q01562

Claims (89)

1. isolating IL-6 antibody that comprises at least one complementarity-determining region with the aminoacid sequence that is selected from SEQ ID NOS:132-137, wherein said antibody combines with people IL-6 or its fragment.
2. the isolated antibody of claim 1, wherein X 1Be A or G, X 2Be S or R, X 3Be H, I, S or Y, X 4Be S or Y, X 5Be S or F, X 6Be F, L, M or T, X 7Be N or E, X 8Be A or T, X 9Be M, C, S or Q, X 10Be Q or C, X 11Be T or Q, X 12Be F, S or T, X 13Be S or P, X 14Be L or M, X 15Be A or I, X 16Be S or P, X 17Be Y or W, X 18Be T, E or Y, X 19Be Y or F, X 20Be P, S, D or Y, X 21Be V or D, X 22Be T or A, X 23Be G or P, X 24Be S, Y, T or N, and X 25Be Y, T, F or I.
3. isolating people's engineered antibody that comprises at least one complementarity-determining region, wherein said antibody combines with people IL-6 or its fragment.
4. isolating IL-6 antibody comprises at least one and has the variable region of heavy chain that is selected from SEQ ID NOS:95,99,103,118,122,126 and 130 aminoacid sequence and/or at least one and have the variable region of light chain that is selected from SEQ ID NOS:93,97,101,116,120,124 and 128 aminoacid sequence.
5. isolating IL-6 antibody that comprises at least one variable region of light chain, described variable region of light chain comprises at least one and is selected from following member:
(a) be selected from SEQ ID NOS:1,3,5,7,9,11,13 and 15 light chain complementarity-determining region 1 (CDRL1) aminoacid sequence;
(b) be selected from SEQ ID NOS:17,19,21,23,25 and 27 CDRL2 aminoacid sequence; With
(c) be selected from SEQ ID NOS:29,31,33,35 and 138 CDRL3 aminoacid sequence.
6. isolating IL-6 antibody that comprises at least one variable region of heavy chain, described variable region of heavy chain comprises at least one and is selected from following member:
(a) be selected from SEQ ID NOS:37,39,41,43,45 and 47 heavy chain complementarity-determining region 1 (CDRH1) aminoacid sequence;
(b) be selected from SEQ ID NOS:49,51,53,55,57,59,61,63,65,67,69,71,73,75,77 and 113 CDRH2 aminoacid sequence; With
(c) be selected from SEQ ID NOS:79,81,83,85,87,89,91 and 114 CDRH3 aminoacid sequence.
7. the isolating IL-6 antibody of the variable region of heavy chain of variable region of light chain that comprises claim 5 and claim 6.
8. the isolating IL-6 antibody of claim 7 further comprises at least one individual framework region that is adjacent at least one complementarity-determining region.
9. the isolating IL-6 antibody of claim 8, wherein at least one individual framework region is selected from SEQ ID NOS:105-112.
10. the isolating IL-6 antibody that comprises at least one variable region of light chain of claim 5, described variable region of light chain comprises:
(a) be selected from SEQ ID NOS:1,3,5,7,9,11,13 and 15 light chain complementarity-determining region 1 (CDRL1) aminoacid sequence;
(b) be selected from SEQ ID NOS:17,19,21,23,25 and 27 CDRL2 aminoacid sequence; (c) be selected from SEQ ID NOS:29,31,33,35 and 138 CDRL3 aminoacid sequence.
11. the isolating IL-6 antibody that comprises at least one variable region of heavy chain of claim 6, described variable region of heavy chain comprises:
(a) be selected from SEQ ID NOS:37,39,41,43,45 and 47 heavy chain complementarity-determining region 1 (CDRH1) aminoacid sequence;
(b) be selected from SEQ ID NOS:49,51,53,55,57,59,61,63,65,67,69,71,73,75,77 and 113 CDRH2 aminoacid sequence; With
(c) be selected from SEQ ID NOS:79,81,83,85,87,89,91 and 114 CDRH3 aminoacid sequence.
12. the isolating IL-6 antibody of the variable region of heavy chain of variable region of light chain that comprises claim 10 and claim 11.
13. the isolated antibody of claim 12, further be included in the FRL4 shown in the FRL3 shown in the FRL2 shown in the people's light chain framework region 1 (FRL1) shown in the SEQ ID NO:105 that is interspersed between the complementarity-determining region, the SEQ IDNO:106, the SEQ ID NO:107, the SEQ ID NO:108, the FRH4 shown in FRH3 shown in the FRH2 shown in the people's heavy chain framework region 1 (FRH1) shown in the SEQ ID NO:109, the SEQ IDNO:110, the SEQ ID NO:111 and the SEQ ID NO:112.
14. the isolated antibody of claim 12, wherein variable region of light chain comprises:
(a) be selected from SEQ ID NOS:1,3,5,7,9,11,13 and 15 CDRL1 aminoacid sequence;
(b) be selected from SEQ ID NOS:17,19,21,23,25 and 27 CDRL2 aminoacid sequence; With
(c) the CDRL3 aminoacid sequence shown in the SEQ ID NO:29.
15. the isolated antibody of claim 12, wherein variable region of light chain comprises:
(a) be selected from SEQ ID NOS:1,3,5,7,9,11,13 and 15 CDRL1 aminoacid sequence;
(b) be selected from SEQ ID NOS:17,19,21,23,25 and 27 CDRL2 aminoacid sequence; With
(c) the CDRL3 aminoacid sequence shown in the SEQ ID NO:35.
16. the isolated antibody of claim 14 or 15 further is included in the FRL4 shown in FRL3 shown in the FRL2 shown in the people's light chain framework region 1 (FRL1) shown in the SEQ ID NO:105 that is interspersed between the CDRLs, the SEQ IDNO:106, the SEQ ID NO:107 and the SEQ ID NO:108.
17. the isolated antibody of claim 12, wherein variable region of heavy chain comprises:
(a) be selected from SEQ ID NOS:37,39,41,43,45 and 47 CDRH1 aminoacid sequence;
(b) be selected from SEQ ID NOS:49,51,53,55,57,59,61,63,65,67,69,71,73,75,77 and 113 CDRH2 aminoacid sequence; With
(c) the CDRH3 aminoacid sequence shown in the SEQ ID NO:87.
18. the isolated antibody of claim 12, wherein variable region of heavy chain comprises:
(a) be selected from SEQ ID NOS:37,39,41,43,45 and 47 CDRH1 aminoacid sequence;
(b) be selected from SEQ ID NOS:49,51,53,55,57,59,61,63,65,67,69,71,73,75,77 and 113 CDRH2 aminoacid sequence; With
(c) the CDRH3 aminoacid sequence shown in the SEQ ID NO:89.
19. the isolated antibody of claim 12, wherein variable region of heavy chain comprises:
(a) be selected from SEQ ID NOS:37,39,41,43,45 and 47 CDRH1 aminoacid sequence;
(b) be selected from SEQ ID NOS:49,51,53,55,57,59,61,63,65,67,69,71,73,75,77 and 113 CDRH2 aminoacid sequence; With
(c) the CDRH3 aminoacid sequence shown in the SEQ ID NO:91.
20. the isolated antibody of claim 12, wherein variable region of heavy chain comprises:
(a) be selected from SEQ ID NOS:37,39,41,43,45 and 47 CDRH1 aminoacid sequence;
(b) be selected from SEQ ID NOS:49,51,53,55,57,59,61,63,65,67,69,71,73,75,77 and 113 CDRH2 aminoacid sequence; With
(c) the CDRH3 aminoacid sequence shown in the SEQ ID NO:114.
21., further be included in the FRH4 shown in FRH3 shown in the FRH2 shown in the people's heavy chain framework region 1 (FRH1) shown in the SEQ ID NO:109 that is interspersed between the CDRHs, the SEQ ID NO:110, the SEQ ID NO:111 and the SEQ ID NO:112 according to each isolated antibody of claim 17-20.
22. the isolated antibody of claim 12, wherein CDRL1 comprises the aminoacid sequence of SEQ ID NO:7, CDRL2 comprises the aminoacid sequence of SEQ ID NO:21, CDRL3 comprises the aminoacid sequence of SEQ ID NO:29, CDRH1 comprises the aminoacid sequence of SEQ ID NO:41, and CDRH2 comprises the aminoacid sequence of SEQ ID NO:75 and the aminoacid sequence that CDRH3 comprises SEQ ID NO:87.
23. the isolated antibody of claim 12, wherein CDRL1 comprises the aminoacid sequence of SEQ ID NO:3, CDRL2 comprises the aminoacid sequence of SEQ ID NO:21, CDRL3 comprises the aminoacid sequence of SEQ ID NO:29, CDRH1 comprises the aminoacid sequence of SEQ ID NO:41, and CDRH2 comprises the aminoacid sequence of SEQ ID NO:75 and the aminoacid sequence that CDRH3 comprises SEQ ID NO:89.
24. the isolated antibody of claim 12, wherein CDRL1 comprises the aminoacid sequence of SEQ ID NO:15, CDRL2 comprises the aminoacid sequence of SEQ ID NO:27, CDRL3 comprises the aminoacid sequence of SEQ ID NO:35, CDRH1 comprises the aminoacid sequence of SEQ ID NO:47, and CDRH2 comprises the aminoacid sequence of SEQ ID NO:61 and the aminoacid sequence that CDRH3 comprises SEQ ID NO:91.
25. the isolated antibody of claim 12, wherein CDRL1 comprises the aminoacid sequence of SEQ ID NO:15, CDRL2 comprises the aminoacid sequence of SEQ ID NO:27, CDRL3 comprises the aminoacid sequence of SEQ ID NO:35, CDRH1 comprises the aminoacid sequence of SEQ ID NO:47, and CDRH2 comprises the aminoacid sequence of SEQ ID NO:57 and the aminoacid sequence that CDRH3 comprises SEQ ID NO:91.
26. the isolated antibody of claim 12, wherein CDRL1 comprises the aminoacid sequence of SEQ ID NO:15, CDRL2 comprises the aminoacid sequence of SEQ ID NO:17, CDRL3 comprises the aminoacid sequence of SEQ ID NO:29, CDRH1 comprises the aminoacid sequence of SEQ ID NO:45, and CDRH2 comprises the aminoacid sequence of SEQ ID NO:59 and the aminoacid sequence that CDRH3 comprises SEQ ID NO:89.
27. the isolated antibody of claim 12, wherein CDRL1 comprises the aminoacid sequence of SEQ ID NO:7, CDRL2 comprises the aminoacid sequence of SEQ ID NO:17, CDRL3 comprises the aminoacid sequence of SEQ ID NO:29, CDRH1 comprises the aminoacid sequence of SEQ ID NO:45, and CDRH2 comprises the aminoacid sequence of SEQ ID NO:77 and the aminoacid sequence that CDRH3 comprises SEQ ID NO:87.
28. isolating IL-6 antibody, comprise light chain complementarity-determining region 1 (CDRL1) aminoacid sequence shown in the SEQ ID NO:3, CDRL2 aminoacid sequence shown in the SEQ ID NO:21, CDRL3 aminoacid sequence shown in the SEQ ID NO:29, heavy chain complementarity-determining region 1 (CDRH1) aminoacid sequence shown in the SEQ ID NO:39, the CDRH3 aminoacid sequence shown in CDRH2 aminoacid sequence shown in the SEQ ID NO:59 and the SEQ ID NO:89.
29. an isolating IL-6 antibody that comprises at least one variable region of light chain, described variable region of light chain comprises:
(a) light chain complementarity-determining region 1 (CDRL1) aminoacid sequence shown in the SEQ ID NO:132;
(b) the CDRL2 aminoacid sequence shown in the SEQ ID NO:133; With
(c) be selected from the CDRL3 aminoacid sequence shown in the SEQ ID NO:134, wherein X 1Be A or G, X 2Be S or R, X 3Be H, I, S or Y, X 4Be S or Y, X 5Be S or F, X 6Be F, L, M or T, X 7Be N or E, X 8Be A or T, X 9Be M, C, S or Q and X 10Be Q or C.
30. an isolating IL-6 antibody that comprises at least one variable region of heavy chain, described variable region of heavy chain comprises:
(a) heavy chain complementarity-determining region 1 (CDRH1) aminoacid sequence shown in the SEQ ID NO:135;
(b) the CDRH2 aminoacid sequence shown in the SEQ ID NO:136; With
(c) the CDRH3 aminoacid sequence shown in the SEQ ID NO:137, wherein X 11Be T or Q, X 12Be F, S or T, X 13Be S or P, X 14Be L or M, X 15Be A or I, X 16Be S or P, X 17Be Y or W, X 18Be T, E or Y, X 19Be Y or F, X 20Be P, S, D or Y, X 21Be V or D, X 22Be T or A, X 23Be G or P, X 24Be S, Y, T or N and X 25Be Y, T, F or I.
31. the isolating IL-6 antibody of the variable region of heavy chain of variable region of light chain that comprises claim 29 and claim 30.
32., further be included in the FRH4 shown in FRH3 shown in the FRH2 shown in the people's heavy chain framework region 1 (FRH1) shown in the FRL4 shown in the FRL3 shown in the FRL2 shown in the people's light chain framework region 1 (FRL1) shown in the SEQ ID NO:105 that is interspersed between the complementarity-determining region, the SEQ ID NO:106, the SEQ ID NO:107, the SEQ ID NO:108, the SEQ ID NO:109, the SEQ ID NO:110, the SEQ ID NO:111 and the SEQ ID NO:112 according to claim 22-28 and 31 each isolating IL-6 antibody.
33. isolating IL-6 antibody that comprises at least one variable region of light chain, described variable region of light chain comprises the FRL4 shown in FRL3 shown in the FRL2 shown in the people's light chain framework region 1 (FRL1) shown in the SEQ ID NO:105, the SEQ IDNO:106, the SEQ ID NO:107 and the SEQ ID NO:108, and the CDR district that is interspersed, this CDR district comprises:
(a) be selected from SEQ ID NOS:1,3,5,7,9,11,13 and 15 CDRL1 aminoacid sequence;
(b) be selected from SEQ ID NOS:17,19,21,23,25 and 27 CDRL2 aminoacid sequence; With
(c) be selected from SEQ ID NOS:29,31,33,35 and 138 CDRL3 aminoacid sequence.
34. isolating IL-6 antibody that comprises at least one variable region of heavy chain, described variable region of heavy chain comprises the FRH4 shown in FRH3 shown in the FRH2 shown in the people's heavy chain framework region 1 (FRH1) shown in the SEQ ID NO:109, the SEQ IDNO:110, the SEQ ID NO:111 and the SEQ ID NO:112, and the CDR district that is interspersed, this CDR district comprises:
(a) be selected from SEQ ID NOS:37,39,41,43,45 and 47 CDRH1 aminoacid sequence;
(b) be selected from SEQ ID NOS:49,51,53,55,57,59,61,63,65,67,69,71,73,75,77 and 113 CDRH2 aminoacid sequence; With
(c) be selected from SEQ ID NOS:79,81,83,85,87,89,91 and 114 CDRH3 aminoacid sequence.
35. the isolating IL-6 antibody of the variable region of heavy chain of variable region of light chain that comprises claim 33 and claim 34.
36. comprising, isolating people or the people's engineered antibody that combines IL-6 with at least a isolating IL-6 antibody competition that comprises at least one variable region of light chain and at least one variable region of heavy chain, described isolating IL-6 antibody be selected from SEQ ID NOS:93 and 95, SEQ ID NOS:97 and 99, SEQ ID NOS:101 and 103, SEQ ID NOS:116 and 118, SEQ ID NOS:120 and 122, SEQ ID NOS:124 and 126 and the light chain and the variable region of heavy chain of SEQ ID NOS:128 and 130.
37. isolating people or people's engineered antibody that combines IL-6 with isolating IL-6 antibody competition according to claim 12.
38. isolating people or people's engineered antibody in a competitive zone in conjunction with the 168-184 amino acids that comprises SEQ ID NO:115.
39. claim 36-38 each antibody, wherein antibody behaviour through engineering approaches.
40. one kind according to each IL-6 antibody of claim 1-39, wherein said antibodies IL-6, have be selected from by surperficial plasmon resonance or Kinexa method measured at least 10 -9M, at least 10 -10M, at least 10 -11M and at least 10 -12M, at least 10 -13M, at least 10 -14M and at least 10 -15At least a avidity of M.
41. according to each IL-6 antibody of claim 1-40, wherein said antibody is regulated at least a activity of at least a IL-6 polypeptide in fact.
42. a coding is at least a according to each the isolated nucleic acid molecule of isolating IL-6 antibody of claim 1-40.
43. isolating nucleic acid carrier that comprises according to the isolated nucleic acid molecule of claim 42.
A 44. protokaryon or eukaryotic host cell that comprises according to the isolated nucleic acid molecule of claim 42.
45. according to the host cell of claim 44, wherein said host cell is be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2,653, SP2/0,293, HeLa, myelomatosis or lymphoma cell or their any derivative, immortalization or transformant at least a.
46. a method that is used to produce at least a IL-6 antibody, be included in external, the body or under the original position condition translation make IL-6 antibody can detect or callable scale reaches according to the nucleic acid molecule of claim 42.
47. one kind comprise at least a according to claim 1-40 each isolating IL-6 antibody and the composition of at least a pharmaceutically acceptable carrier or thinner.
48., further comprise at least a compound or the polypeptide that are selected from detectable mark or reporter molecule, TNF antagonist, anti-infective, cardiovascular (CV) system medicine, central nervous system (CNS) medicine, autonomous nervous system (ANS) medicine, respiratory tract medicine, stomach and intestine (GI) road medicine, hormone drug, the medicine that is used for water or electrolyte balance, hematologic drug, antitumor agent, immunomodulator, medicament for the eyes, ear medicine or nose medicine, topical drug, nutritional drugs, cytokine and cytokine antagonist according to the composition of claim 47.
49. a specific specificity is bonded to less a kind of according to each the antiidiotypic antibody or the fragment of IL-6 antibody of claim 1-40.
50. one kind is used for diagnosing or treat the method that cell, tissue, organ or animal IL-6 have related disorders, comprises at least aly contacting with described cell, tissue, organ or animal or being applied to described cell, tissue, organ or animal according to each the composition of antibody of claim 1-40 what comprise significant quantity.
51. according to the method for claim 50, wherein the relevant illness of IL-6 is selected from rheumatoid arthritis, osteoarthritis, osteolysis, orthopaedic implants aseptic loosening, systemic lupus erythematosus, lupus erythematosus,cutaneous, lupus nephritis, type ii diabetes, chronic obstructive pulmonary disease and renal cell carcinoma.
52. according to the method for claim 50, wherein said significant quantity is the described cell of about 0.001-50mg/kg, tissue, organ or animal.
53. according to the method for claim 50, wherein by being selected from parenteral, subcutaneous, intramuscular, intravenously, intraarticular, in the segmental bronchus, in the abdomen, in the capsule, in the cartilage, in the chamber, in the body cavity, in the cerebellum, Intraventricular, colonic, in the uterine cervix, in the stomach, in the liver, in the cardiac muscle, in the bone, in the pelvis, in the pericardium, intraperitoneal, in the pleura, in the prostate gland, in the lung, internal rectum, in the kidney, in the retina, in the backbone, in the synovial membrane, in the thoracic cavity, intrauterine, intravesical, in the damaging part, inject, vagina, rectum, the oral cavity contains clothes, the hypogloeeis, in the nose with carry out described the contact or described using through at least a mode of skin.
54. method according to claim 50, before further being included in described contact or using, simultaneously or use at least a at least a compound of significant quantity or the composition of polypeptide of comprising afterwards, described at least a compound or polypeptide are selected from detectable mark or reporter molecule, anti-infective, cardiovascular (CV) system medicine, central nervous system (CNS) medicine, autonomous nervous system (ANS) medicine, the respiratory tract medicine, stomach and intestine (GI) road medicine, hormone drug, the medicine that is used for water or electrolyte balance, hematologic drug, antitumor agent, immunomodulator, medicament for the eyes, ear medicine or nose medicine, topical drug, nutritional drugs, cytokine and cytokine antagonist.
55. one kind comprises each the medical treatment device of IL-6 antibody according to claim 1-40, wherein said device is adapted to pass through and is selected from parenteral, subcutaneous, intramuscular, intravenously, intraarticular, in the segmental bronchus, in the abdomen, in the capsule, in the cartilage, in the chamber, in the body cavity, in the cerebellum, Intraventricular, colonic, in the uterine cervix, in the stomach, in the liver, in the cardiac muscle, in the bone, in the pelvis, in the pericardium, intraperitoneal, in the pleura, in the prostate gland, in the lung, internal rectum, in the kidney, in the retina, in the backbone, in the synovial membrane, in the thoracic cavity, intrauterine, intravesical, in the damaging part, inject, vagina, rectum, the oral cavity contains clothes, the hypogloeeis, nose at least a mode interior and through skin contacts or uses described IL-6 antibody.
56. a product that is used for people's medicine or diagnostic use, comprise wrapping material and contain solution or lyophilized form according to each the container of IL-6 antibody of claim 1-40.
57. the product of claim 56, wherein said container is a parenteral, subcutaneous, intramuscular, intravenously, intraarticular, in the segmental bronchus, in the abdomen, in the capsule, in the cartilage, in the chamber, in the body cavity, in the cerebellum, Intraventricular, colonic, in the uterine cervix, in the stomach, in the liver, in the cardiac muscle, in the bone, in the pelvis, in the pericardium, intraperitoneal, in the pleura, in the prostate gland, in the lung, internal rectum, in the kidney, in the retina, in the backbone, in the synovial membrane, in the thoracic cavity, intrauterine, intravesical, in the damaging part, inject, vagina, rectum, the oral cavity contains clothes, the hypogloeeis, in the nose or through the integral part of skin drug delivery systems or system.
58. a generation comprises host cell or transgenic animal or the transgenic plant or the vegetable cell that can reach described antibody with recyclable scale is provided according to each the method for isolating IL-6 antibody of claim 1-40.
59. IL-6 antibody that produces according to the method for claim 58.
60. the isolated nucleic acid molecule of the IL-6 antibody of encoding comprises at least one variable region of light chain, described variable region of light chain comprises and is selected from least one following member:
(a) be selected from SEQ ED NOS:2,4,6,8,10,12,14 and 16 light chain complementarity-determining region 1 (CDRL1) nucleotide sequence;
(b) be selected from SEQ ID NOS:18,20,22,24,26 and 28 CDRL2 nucleotide sequence; With
(c) be selected from SEQ TD NOS:30,32,34 and 36 CDRL3 nucleotide sequence.
61. the isolated nucleic acid molecule of the IL-6 antibody of encoding comprises at least one variable region of heavy chain, described variable region of heavy chain comprises and is selected from least one following member:
(a) be selected from SEQ ID NOS:38,40,42,44,46 and 48 heavy chain complementarity-determining region 1 (CDRH1) nucleotide sequence;
(b) be selected from SEQ ID NOS:50,52,54,56,58,60,62,64,66,68,70,72,74,76 and 78 CDRH2 nucleotide sequence; With
(c) be selected from SEQ ID NOS:80,82,84,86,88,90 and 92 CDRH3 nucleotide sequence.
62. an encoded packets contains the isolated nucleic acid molecule of IL-6 antibody that right requires the weight chain variable region nucleotide sequence of 60 light chain variable region nucleotide sequence and claim 61.
63. the isolated nucleic acid molecule of the IL-6 antibody of encoding comprises at least one variable region of light chain, described variable region of light chain comprises at least one and is selected from following member:
(a) be selected from SEQ ID NOS:2,4,6,8,10,12,14 and 16 light chain complementarity-determining region 1 (CDRL1) nucleotide sequence;
(b) be selected from SEQ ID NOS:18,20,22,24,26 and 28 CDRL2 nucleotide sequence; With
(c) be selected from SEQ ID NOS:30,32,34 and 36 CDRL3 nucleotide sequence.
64. the isolated nucleic acid molecule of the IL-6 antibody of encoding comprises at least one variable region of heavy chain, described variable region of heavy chain comprises:
(a) be selected from SEQ ID NOS:38,40,42,44,46 and 48 heavy chain complementarity-determining region 1 (CDRH1) nucleotide sequence;
(b) be selected from SEQ ID NOS:50,52,54,56,58,60,62,64,66,68,70,72,74,76 and 78 CDRH2 nucleotide sequence; With
(c) be selected from SEQ ID NOS:80,82,84,86,88,90 and 92 CDRH3 nucleotide sequence.
65. the isolated nucleic acid molecule of the coding IL-6 antibody of the weight chain variable region nucleotide sequence of light chain variable region nucleotide sequence that comprises claim 63 and claim 64.
66. the isolated nucleic acid molecule of claim 65 further is included in the nucleotide sequence of FRH3 shown in the people's heavy chain framework region 1 (FRH1) shown in the FRL3 shown in the people's light chain framework region 1 (FRL1) shown in the coding SEQ ID NO:105 that is interspersed between the complementarity-determining region nucleotide sequence, the FRL2 shown in the SEQ ID NO:106, the SEQ ID NO:107, the FRL4 shown in the SEQ ID NO:108, the SEQ ID NO:109, the FRH2 shown in the SEQ ID NO:110, the SEQ ID NO:111 and the FRH4 shown in the SEQ ID NO:112.
67. the isolated nucleic acid molecule of claim 63, wherein variable region of light chain comprises:
(a) be selected from SEQ ID NOS:2,4,6,8,10,12,14 and 16 CDRL1 nucleotide sequence;
(b) be selected from SEQ ID NOS:18,20,22,24,26 and 28 CDRL2 nucleotide sequence; With
(c) the CDRL3 nucleotide sequence shown in the SEQ ID NO:30.
68. the isolated nucleic acid molecule of claim 63, wherein variable region of light chain comprises:
(a) be selected from SEQ ID NOS:2,4,6,8,10,12,14 and 16 CDRL1 nucleotide sequence;
(b) be selected from SEQ ID NOS:18,20,22,24,26 and 28 CDRL2 nucleotide sequence; With
(c) the CDRL3 nucleotide sequence shown in the SEQ ID NO:36.
69. the isolated nucleic acid molecule of claim 64, wherein variable region of heavy chain comprises:
(a) be selected from SEQ ID NOS:38,40,42,44,46 and 48 CDRH1 nucleotide sequence;
(b) be selected from SEQ ID NOS:50,52,54,56,58,60,62,64,66,68,70,72,74,76 and 78 CDRH2 nucleotide sequence; With
(c) the CDRH3 nucleotide sequence shown in the SEQ ID NO:86.
70. the isolated nucleic acid molecule of claim 64, wherein variable region of heavy chain comprises:
(a) be selected from SEQ ID NOS:38,40,42,44,46 and 48 CDRH1 nucleotide sequence;
(b) be selected from SEQ ID NOS:50,52,54,56,58,60,62,64,66,68,70,72,74,76 and 78 CDRH2 nucleotide sequence; With
(c) the CDRH3 nucleotide sequence shown in the SEQ ID NO:88.
71. the isolated nucleic acid molecule of claim 64, wherein variable region of heavy chain comprises:
(a) be selected from SEQ ID NOS:38,40,42,44,46 and 48 CDRH1 nucleotide sequence;
(b) be selected from SEQ ID NOS:50,52,54,56,58,60,62,64,66,68,70,72,74,76 and 78 CDRH2 nucleotide sequence; With
(c) the CDRH3 nucleotide sequence shown in the SEQ ID NO:90.
72. the isolated nucleic acid molecule of claim 64, wherein variable region of heavy chain comprises:
(a) be selected from SEQ ID NOS:38,40,42,44,46 and 48 CDRH1 nucleotide sequence;
(b) be selected from SEQ ID NOS:50,52,54,56,58,60,62,64,66,68,70,72,74,76 and 78 CDRH2 nucleotide sequence; With
(c) the CDRH3 nucleotide sequence shown in the SEQ ID NO:92.
73. the isolated nucleic acid molecule of claim 65, comprise the CDRL1 nucleotide sequence shown in the SEQ ID NO:8, CDRL2 nucleotide sequence shown in the SEQ ID NO:22, CDRL3 nucleotide sequence shown in the SEQID NO:30, CDRH1 nucleotide sequence shown in the SEQ ID NO:42, the CDRH3 nucleotide sequence shown in CDRH2 nucleotide sequence shown in the SEQ ID NO:76 and the SEQ ID NO:88.
74. the isolated nucleic acid molecule of claim 65, comprise the CDRL1 nucleotide sequence shown in the SEQ ID NO:4, CDRL2 nucleotide sequence shown in the SEQ ID NO:22, CDRL3 nucleotide sequence shown in the SEQID NO:30, CDRH1 nucleotide sequence shown in the SEQ ID NO:40, the CDRH3 nucleotide sequence shown in CDRH2 nucleotide sequence shown in the SEQ ID NO:60 and the SEQ ID NO:90.
75. the isolated nucleic acid molecule of claim 65, comprise the CDRL1 nucleotide sequence shown in the SEQ ID NO:4, CDRL2 nucleotide sequence shown in the SEQ ID NO:22, CDRL3 nucleotide sequence shown in the SEQID NO:30, CDRH1 nucleotide sequence shown in the SEQ ID NO:42, the CDRH3 nucleotide sequence shown in CDRH2 nucleotide sequence shown in the SEQ ID NO:76 and the SEQ ID NO:90.
76. the isolated nucleic acid molecule of claim 65, comprise the CDRL1 nucleotide sequence shown in the SEQ ID NO:16, CDRL2 nucleotide sequence shown in the SEQ ID NO:28, CDRL3 nucleotide sequence shown in the SEQID NO:36, CDRH1 nucleotide sequence shown in the SEQ ID NO:48, the CDRH3 nucleotide sequence shown in CDRH2 nucleotide sequence shown in the SEQ ID NO:62 and the SEQ ID NO:92.
77. the isolated nucleic acid molecule of claim 65, comprise the CDRL1 nucleotide sequence shown in the SEQ ID NO:16, CDRL2 nucleotide sequence shown in the SEQ ID NO:28, CDRL3 nucleotide sequence shown in the SEQID NO:36, the CDRH1 nucleotide sequence of SEQ ID NO:48, the CDRH3 nucleotide sequence shown in CDRH2 nucleotide sequence shown in the SEQ ID NO:58 and the SEQ ID NO:92.
78. the isolated nucleic acid molecule of claim 65, comprise the CDRL1 nucleotide sequence shown in the SEQ ID NO:16, CDRL2 nucleotide sequence shown in the SEQ ID NO:18, CDRL3 nucleotide sequence shown in the SEQID NO:30, CDRH1 nucleotide sequence shown in the SEQ ID NO:46, the CDRH3 nucleotide sequence shown in CDRH2 nucleotide sequence shown in the SEQ ID NO:60 and the SEQ ID NO:90.
79. the isolated nucleic acid molecule of claim 65, comprise the CDRL1 nucleotide sequence shown in the SEQ ID NO:8, CDRL2 nucleotide sequence shown in the SEQ ID NO:18, CDRL3 nucleotide sequence shown in the SEQID NO:30, CDRH1 nucleotide sequence shown in the SEQ ID NO:46, the CDRH3 nucleotide sequence shown in CDRH2 nucleotide sequence shown in the SEQ ID NO:78 and the SEQ ID NO:88.
80. one kind according to each IL-6 antibody of claim 1-39, wherein said antibody has about 2.7 * 10 -11M or littler EC 50Value.
81. 0 antibody, wherein EC according to Claim 8 50Value is for about 2.7 * 10 -12Or it is littler.
82. an isolating IL-6 antibody comprises the light chain variable region amino acid sequence of SEQ ID NO:93 and the weight chain variable region amino acid sequence of SEQ ID NO:95.
83. an isolating IL-6 antibody comprises the light chain variable region amino acid sequence of SEQ ID NO:97 and the weight chain variable region amino acid sequence of SEQ ID NO:99.
84. an isolating IL-6 antibody comprises the light chain variable region amino acid sequence of SEQ ID NO:101 and the weight chain variable region amino acid sequence of SEQ ID NO:103.
85. an isolating IL-6 antibody comprises the light chain variable region amino acid sequence of SEQ ID NO:116 and the weight chain variable region amino acid sequence of SEQ ID NO:118.
86. an isolating IL-6 antibody comprises the light chain variable region amino acid sequence of SEQ ID NO:120 and the weight chain variable region amino acid sequence of SEQ ID NO:122.
87. an isolating IL-6 antibody comprises the light chain variable region amino acid sequence of SEQ ID NO:124 and the weight chain variable region amino acid sequence of SEQ ID NO:126.
88. an isolating IL-6 antibody comprises the light chain variable region amino acid sequence of SEQ ID NO:128 and the weight chain variable region amino acid sequence of SEQ ID NO:130.
89. an isolating IL-6 antibody is included as the coded light chain variable region amino acid sequence of one of SEQ ID NOS:94, nucleotide sequence of 98,102,117,121,125 and 129 and is one of SEQ ID NOS:96,100,104,119,123,127 and 131 nucleotide sequence coded weight chain variable region amino acid sequence.
90. the described any invention of the application.
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